Use of saposin-related proteins for preventing and treating obesity, diabetes and/or metabolic syndrome

ABSTRACT

The present invention discloses Saposin-related homologous proteins regulating the energy homeostasis and the metabolism of triglycerides, and polynucleotides, which identify and encode the proteins disclosed in this invention. The invention also relates to the use of these sequences in the diagnosis, study, prevention, and treatment of metabolic diseases and disorders.

This invention relates to the use of saposin-related proteins, to theuse of polynucleotides encoding these, and to the use of effectorsthereof in the diagnosis, study, prevention, and treatment of obesityand/or diabetes mellitus and/or metabolic syndrome.

Many human proteins serve as pharmaceutically active compounds. Severalclasses of human proteins that serve as such active compounds includehormones, cytokines, cell growth factors, and cell differentiationfactors. Most proteins that can be used as a pharmaceutically activecompound fall within the family of secreted proteins. Secreted proteinsare generally produced within cells at rough endoplasmic reticulum, arethen exported to the golgi complex, and then move to secretory vesiclesor granules, where they are secreted to the exterior of the cell viaexocytosis. Examples for commercially used secreted proteins are humaninsulin, thrombolytic agents, interferons, interleukins, colonystimulating factors, human growth hormone, transforming growth factorbeta, tissue plasminogen activator, erythropoeitin, and various otherproteins. Receptors of secreted proteins, which are membrane-boundproteins, also have potential as therapeutic or diagnostic agents.

It is, therefore, important for developing new pharmaceutical compoundsto identify secreted proteins that can be tested for activity in avariety of animal models. Thus, in light of the pervasive role ofsecreted proteins in human physiology, a need exists for identifying andcharacterizing novel functions for human secreted proteins and the genesthat encode them. This knowledge will allow one to detect, to treat, andto prevent medical diseases, disorders, and/or conditions by usingsecreted proteins or the genes that encode them.

Obesity is one of the most prevalent metabolic disorders in the world.It is still a poorly understood human disease that becomes as a majorhealth problem more and more relevant for western society. Obesity isdefined as a body weight more than 20% in excess of the ideal bodyweight, frequently resulting in a significant impairment of health.Obesity may be measured by body mass index, an indicator of adiposity orfatness. Further parameters for defining obesity are waistcircumferences, skinfold thickness and bioimpedance. It is associatedwith an increased risk for cardiovascular disease, hypertension,diabetes mellitus type II, hyperlipidaemia and an increased mortalityrate.

Obesity is influenced by genetic, metabolic, biochemical, psychological,and behavioral factors and can be caused by different reasons such asnon-insulin dependent diabetes, increase in triglycerides, increase incarbohydrate bound energy and low energy expenditure. As such, it is acomplex disorder that must be addressed on several fronts to achievelasting positive clinical outcome. Since obesity is not to be consideredas a single disorder but as a heterogeneous group of conditions with(potential) multiple causes, it is also characterized by elevatedfasting plasma insulin and an exaggerated insulin response to oralglucose intake (Koltermann J., (1980) Clin. Invest 65, 1272-1284). Aclear involvement of obesity in type 2 diabetes mellitus can beconfirmed (Kopelman P. G., (2000) Nature 404, 635-643).

Triglycerides and glycogen are used as the body's fuel energy storage.Glycogen is a large branched polymer of glucose residues that is mainlystored in liver and muscle cells. Glycogen synthesis and degradation iscentral to the control of the blood glucose level. Triglycerides arestored in the cytoplasm of adipocytes. Adipocytes are specialized forthe synthesis, storage and mobilization of triglycerides. The glycogenand triglyceride metabolism is highly regulated and their interplay isessential for the energy homeostasis of the body. A high glucose levelin the adipocytes results in the synthesis of triglycerides as fuelstore. A low intracellular glucose level leads to a release of fattyacids, which can be used as substrates for the beta-oxidation togenerate energy. Glycogen levels in cells are more variable thantriglyceride levels because the turnover of glycogen is higher.Triglycerides are used as long term energy donors once the glycogenstores run low.

Pancreatic beta-cells secrete insulin in response to blood glucoselevels. Insulin amongst other hormones plays a key role in theregulation of the fuel metabolism. Insulin leads to the storage ofglycogen and triglycerides and to the synthesis of proteins. The entryof glucose into muscles and adipose cells is stimulated by insulin. Inpatients who suffer from diabetes mellitus type I or LADA (latentautoimmue diabetes in adults (Pozzilli & Di Mario, 2001, Diabetes Care.8:1460-67) beta-cells are being destroyed due to autoimmune attack. Theamount of insulin produced by the remaining pancreatic islet cells istoo low, resulting in elevated blood glucose levels (hyperglycemia). Indiabetes type II liver and muscle cells loose their ability to respondto normal blood insulin levels (insulin resistance). High blood glucoselevels (and also high blood lipid levels) in turn lead to an impairmentof beta-cell function and to an increase in beta-cell apoptosis. It isinteresting to note that the rate of beta-cell neogenesis does notappear to change in type II diabetics (Butler et al., 2003 supra), thuscausing a reduction in total beta-cell mass over time. Eventually theapplication of exogenous insulin becomes necessary in type II diabetics.

Improving metabolic parameters such as blood sugar and blood lipidlevels (e.g. through dietary changes, exercise, medication orcombinations thereof) before beta-cell mass has fallen below a criticalthreshold leads to a relatively rapid restoration of beta-cell function.However, after such a treatment the pancreatic endocrine function wouldremain impaired due to the only slightly increased regeneration rate.

In type I diabetics, where beta-cells are being destroyed by autoimmuneattack, treatments have been devised which modulate the immune systemand may be able to stop or strongly reduce islet destruction (Raz etal., 2001, Lancet 358: 1749-1753; Chatenoud et al., 2003, Nat RevImmunol. 3: 123-132; Homann et al., Immunity. 2002, 3:403-15). However,due to the relatively slow regeneration of human beta-cells suchtreatments can only be successful if they are combined with agents thatcan stimulate beta-cell regeneration.

Diabetes is a very disabling disease, because today's commonanti-diabetic drugs do not control blood sugar levels well enough tocompletely prevent the occurrence of high and low blood sugar levels.Out of range blood sugar levels are toxic and cause long-termcomplications like for example renopathy, retinopathy, neuropathy andperipheral vascular disease. There is also a host of related conditions,such as obesity, hypertension, heart disease and hyperlipidemia, forwhich persons with diabetes are substantially at risk.

Apart from the impaired quality of life for the patients, the treatmentof diabetes and its long term complications presents an enormousfinancial burden to our healthcare systems with rising tendency. Thus,for the treatment of, type I and type II diabetes as well as for latentautoimmune diabetes in adults (LADA) there is a strong need in the artto identify factors that induce regeneration of pancreatic insulinproducing beta-cells. These factors could restore normal function of theendocrine pancreas once its function is impaired or event could preventthe development or progression of diabetes type I, diabetes type II, orLADA.

The concept of ‘metabolic syndrome’ (syndrome x, insulin-resistancesyndrome, deadly quartet) was first described 1966 by Camus andreintroduced 1988 by Reaven (Camus J P, 1966, Rev Rhum Mal Osteoartic33(1):10-14; Reaven et al. 1988, Diabetes, 37(12):1595-1607). Todaymetabolic syndrome is commonly defined as clustering of cardiovascularrisk factors like hypertension, abdominal obesity, high blood levels oftriglycerides and fasting glucose as well as low blood levels of HDLcholesterol. Insulin resistance greatly increases the risk of developingthe metabolic syndrome (Reaven, 2002, Circulation 106(3): 286-288). Themetabolic syndrome often precedes the development of type II diabetesand cardiovascular disease (McCook, 2002, JAMA 288: 2709-2716). Thecontrol of blood lipid levels and blood glucose levels is the essentialfor the treatment of the metabolic syndrome (see, for example,Santomauro A. T. et al., (1999) Diabetes, 48(9):1836-1841).

The molecular factors regulating food intake and body weight balance areincompletely understood. Even if several candidate genes have beendescribed which are supposed to influence the homeostatic system(s) thatregulate body mass/weight, like leptin or the peroxisomeproliferator-activated receptor-gamma co-activator, the distinctmolecular mechanisms and/or molecules influencing obesity or bodyweight/body mass regulations are not known.

Secreted proteins are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize novel functions for secreted proteins. Thepresent invention advances the state of the art by providing previouslyunknown functions for a human secreted protein.

The technical problem underlying the present invention was to providefor means and methods for modulating (pathological) metabolic conditionsinfluencing body-weight regulation and/or energy homeostatic circuits.Also the technical problem was to provide methods for treating diabetes.The solution to said technical problems is achieved by providing theembodiments characterized in the claims.

Accordingly, the present invention relates to genes encoding secretedproteins with novel functions in body-weight regulation, energyhomeostasis, metabolism, obesity and regeneration processes. The presentinvention discloses a specific gene involved in the regulation ofbody-weight, energy homeostasis, metabolism, and obesity, as well asrelated diseases such as diabetes mellitus, eating disorder, cachexia,hypertension, coronary heart disease, hypercholesterolemia(dyslipidemia), and/or gallstones. The present invention describessaposin-related genes, their homologous genes and proteins encodedthereby, in particular human saposin-related genes and proteins (alsoreferred to as prosaposin, variant Gaucher disease and variantmetachromatic leukodystrophy; PSAP; human pulmonarysurfactant-associated protein B (SFTPB); human hypothetical proteinFLJ40379), respectively, as being involved in the conditions mentionedabove. We disclose a novel use of prosaposin to stimulate the formationor regeneration of insulin-producing beta cells and thus, a use in thetreatment and prevention of diseases going along with (e.g. caused by,associated with or accompanied by) impaired beta-cell function, forexample but not limited to diabetes mellitus. More particularly, thediseases are diabetes type I, diabetes type II or LADA.

So far, it has not been described that the proteins of the invention andhomologous proteins are involved in the regulation of energy homeostasisand body-weight regulation or in regeneration processes and relateddisorders, and thus, no functions in metabolic diseases and dysfunctionsand other diseases as listed above have been discussed.

In this invention we refer to the proteins encoded by DrosophilaSaposin-related genes and homologous proteins, preferably human andmurine homologous polypeptides or proteins or sequences encoding theseproteins as saposin-related or as proteins of the invention.

The present invention discloses that saposin-related proteins areregulating the energy homeostasis and fat metabolism especially themetabolism and storage of triglycerides and glycogen, andpolynucleotides, which identify and encode the proteins disclosed inthis invention. The invention also relates to vectors, host cells,antibodies, and recombinant methods for producing the polypeptides andpolynucleotides of the invention. The invention also relates to the useof these polynucleotides, polypeptides and effectors thereof in thediagnosis, study, prevention, and treatment of metabolic diseases ordysfunctions, for example, but not limited to, metabolic syndrome,obesity, diabetes mellitus, eating disorder, cachexia, hypertension,coronary heart disease, hypercholesterolemia (dyslipidemia), and/orgallstones.

The present invention is also based on the finding that prosaposinstimulates the differentation of insulin producing cells from stem cellsin vitro. Thus, a therapeutically effective amount of prosaposin productmay be administered to promote the regeneration of pancreatic beta-cellsor to promote the formation of insulin-producing cells from stem cellsor progenitor cells in vitro or in vivo. The present invention furtherrelates to applications in the medical field that directly arise fromthe method of the invention. Additionally, the present invention relatesto applications for the identification and characterization of compoundswith therapeutic medical effects or toxicological effects that directlyarise from the method of the invention.

Saposin-related homologous proteins and nucleic acid molecules codingtherefore are obtainable from insect or vertebrate species, e.g. mammalsor birds. Particularly preferred are nucleic acids encoding the humansaposin-related homologs and the proteins encoded thereby (in particularhuman prosaposin [PSAP], human pulmonary surfactant-associated protein B[SFTPB], and/or human hypothetical protein FLJ40379).

The glycoprotein Prosaposin (PSAP) is a precursor for four saposins (A,B, C, and D) found in different cellular locations (Morimoto S. et al.,(1989) Proc. Nat. Acad. Sci. 86: 3389-3393). Saposins are smalllysosomal proteins that serve as activators of various lysosomallipid-degrading enzymes (Munford R. S. et al., (1995) J. Lipid Res. 36:1653-1663). Prosaposin occurs in various kinds of human secretory fluidssuch as cerebrospinal fluid, semen, milk, pancreatic juice, and bile.Prosaposin is postulated to mediate neurotrophic signaling eventscapable of inducing neural differentiation and preventing cell death.

A mutant mouse line in which the sphingolipid activator protein gene hasbeen inactivated by homologous recombination technology showed complexclinical, pathologic, and biochemical abnormalities similar to those ofhuman patients with total saposin deficiency (Fujita N. et al., (1996)Hum Mol Genet. 5: 711-725). The main pathology in the brain of affectedSap −/− mice was hypomyelination and storage of ceramides andgangliosides. Ceramide storage occurred also in brain, liver, andkidney, and ceramide catabolism is abnormally slow in fibroblasts.Examination of reproductive organs in prosaposin homozygous mutant malesshows several abnormalities, indicating that prosaposin is also involvedin the development and maintenance of male reproductive organs (MoralesC. R. et al., (2000) J Androl 21: 765-775).

Pulmonary surfactant is a lipid-rich material that prevents lungcollapse by lowering surface tension at the air-liquid interface in thealveoli of lung. It is composed primarily of phospholipids, cholesteroland proteins, including four surfactant associated proteins, twocollagenous, carbohydrate-binding glycoproteins (PSP-A and PSP-D), andtwo small hydrophobic proteins (PSP-B and PSP-C). Pulmonary surfactantproteins are involved in respiration, and promote alveolar stability bylowering the surface tension at the air-liquid interface in theperipheral air spaces. PSP-B is a clinically important, developmentallyregulated gene. Deficiency of PSPB B was demonstrated in congenitalalveolar proteinosis (Nogee et al., (1993) N Engl J. Med. 328: 406-410).

The invention particularly relates to a nucleic acid molecule encoding apolypeptide contributing to regulating the energy homeostasis and themetabolism of triglycerides and glycogen and regeneration processes,wherein said nucleic acid molecule comprises

-   (a) the nucleotide sequence of the Drosophila saposin-related gene    or a mammalian, e.g. human saposin-related homolog (in particular    human PSAP, SFTPB and/or FLJ40379), and/or a sequence complementary    thereto,-   (b) a nucleotide sequence which hybridizes at 50° C. in a solution    containing 1×SSC and 0.1% SDS to a sequence of (a),-   (c) a sequence corresponding to the sequences of (a) or (b) within    the degeneration of the genetic code,-   (d) a sequence which encodes a polypeptide which is at least 85%,    preferably at least 90%, more preferably at least 95%, more    preferably at least 98% and up to 99.6% identical to the amino acid    sequences of a saposin-related protein, preferably a mammalian, e.g.    human saposin-related homolog (in particular PSAP, SFTPB, and/or    FLJ40379),-   (e) a sequence which differs from the nucleic acid molecule of (a)    to (d) by mutation and wherein said mutation causes an alteration,    deletion, duplication and/or premature stop in the encoded    polypeptide or-   (f) a partial sequence of any of the nucleotide sequences of (a)    to (e) having a length of 15-25 bases, preferably 25-35 bases, more    preferably 35-50 bases and most preferably at least 50 bases.

The present invention relates to genes with novel functions inregeneration processes, body-weight regulation, energy homeostasis,metabolism, and obesity, fragments of said genes, polypeptides encodedby said genes or fragments thereof, and effectors e.g. antibodies,biologically active nucleic acids, such as antisense molecules, RNAimolecules or ribozymes, aptamers, peptides or low-molecular weightorganic compounds recognizing said polynucleotides or polypeptides.

Further, the present invention relates to compositions for modulatingadipogenis, for modulating, e.g. stimulating pancreatic developmentand/or for the regeneration of pancreatic cells or tissues, e.g. cellshaving exocrinous functions, such as acinar cells, centroacinar cellsand/or ductal cells, and/or cells having endocrinous functions,particularly cells in Langerhans islets such as alpha-, beta-, delta-,and/or PP-cells, more particularly beta-cells.

The present invention relates to new methods for stimulating and/orinducing the differentiation of progenitor cells, e.g. stem cells intoinsulin-producing cells or for promoting the protection, survival and/orregeneration of insulin producing cells using a prosaposin productand/or a modulator/effector thereof that influences, particularlyincreases the expression level or function of a prosaposin proteinproduct.

Thus, the present invention provides methods for treating patientssuffering from a disease caused by, associated with, and/or acompaniedby functionally impaired and/or reduced numbers of pancreatic isletcells, particularly insulin producing beta-cells, by administering atherapeutically effective amount of a prosaposin product or a compoundthat influences the prosaposin expression level or function. Functionalimpairment or loss of pancreatic islet cells may be due to e.g.autoimmune attack such as in diabetes type I or LADA, and/or due to celldegeneration such as in progressed diabetes type II. The methods of thepresent invention may also be used to treat patients at risk to developdegeneration of insulin producing beta-cells to prevent the start orprogress of such process.

The prosaposin product or the effector/modulator thereof may beadministered e.g. as a pharmaceutical composition, via implantation ofprosaposin product expressing cells and/or via gene therapy.

Further, the invention relates to cell preparations comprisingprosaposin-treated insulin producing cells or prosaposin expressingcells.

Numerous additional aspects and advantages of the invention will becomeapparent to those skilled in the art upon consideration of the followingdescription of the Figures and detailed description of the inventionwhich describes presently preferred embodiments thereof.

The ability to manipulate and screen the genomes of model organisms suchas the fly Drosophila melanogaster provides a powerful tool to analyzebiological and biochemical processes that have direct relevance to morecomplex vertebrate organisms due to significant evolutionaryconservation of genes, cellular processes, and pathways (see, forexample, Adams M. D. et al., (2000) Science 287: 2185-2195).Identification of novel gene functions in model organisms can directlycontribute to the elucidation of correlative pathways in mammals(humans) and of methods of modulating them. A correlation between apathology model (such as changes in triglyceride levels as indicationfor metabolic syndrome including obesity) and the modified expression ofa fly gene can identify the association of the human ortholog with theparticular human disease.

A forward genetic screen is performed in fly displaying a mutantphenotype due to misexpression of a known gene (see, Johnston Nat RevGenet 3: 176-188 (2002); Rorth P., (1996) Proc Natl Acad Sci USA 93:12418-12422). In this invention, we have used a genetic screen toidentify mutations of Saposin-related homologous genes that causechanges in the body weight, which are reflected by a significant changeof triglyceride levels. Additionally glycogen levels are analysed.

One resource for screening was a Drosophila melanogaster stockcollection of EP-lines. The P-vector of this collection hasGal4-UAS-binding sites fused to a basal promoter that can transcribeadjacent genomic Drosophila sequences upon binding of Gal4 to UAS-sites(Brand & Perrimon (1993) Development 118: 401-415; Rorth P., supra).This enables the EP-line collection for overexpression of endogenousflanking gene sequences. In addition, without activation of theUAS-sites, integration of the EP-element into the gene is likely tocause a reduction of gene activity, and allows determining its functionby evaluating the loss-of-function phenotype.

To isolate genes with a function in energy homeostasis, several thousandEP-lines were tested for their triglyceride/glycogen content after aprolonged feeding period (see Examples for more detail). Lines withsignificantly changed triglyceride/glycogen content were selected aspositive candidates for further analysis. The change oftriglyceride/glycogen content due to the loss of a gene functionsuggests gene activities in energy homeostasis in a dose dependentmanner that control the amount of energy stored as triglycerides orglycogen.

In this invention, the content of triglycerides and glycogen of a poolof flies with the same genotype was analyzed after feeding for six daysusing a triglyceride and a glycogen assay. Male flies homozygous for theintegration of vectors for Drosophila lines HD-EP(3)36824 were analyzedin assays measuring the triglyceride/glycogen contents of these flies(illustrated in more detail in the Examples section). The results of thetriglyceride/glycogen content analysis are shown in FIG. 1.

Genomic DNA sequences were isolated that are localized directly adjacentto the EP vector (herein HD-EP(3)36824) integration. Using thoseisolated genomic sequences public databases like Berkeley DrosophilaGenome Project (GadFly; see also FlyBase (1999) Nucleic Acids Research27:85-88) were screened thereby identifying the integration site of thevectors, and the corresponding gene, described in more detail in theExamples section. The molecular organization of the gene is shown inFIG. 2.

The Drosophila genes and proteins encoded thereby with functions in theregulation of triglyceride metabolism were further analysed in publiclyavailable sequence databases (see Eamples for more detail) and mammalianhomologs were identified (see FIG. 3).

In addition, we identified prosaposin (PSAP) as secreted factorexpressed in developing mouse pancreas, as described in more detail inthe Examples section.

The function of the mammalian homologs in energy homeostasis was furthervalidated in this invention by analyzing the expression of thetranscripts in different tissues and by analyzing the role in adipocytedifferentiation.

Expression profiling studies (see Examples for more detail) confirm theparticular relevance of the protein of the invention as regulators ofenergy metabolism in mammals. Transcripts of prosaposin are found inseveral tissues of mammals with high expression levels in brain tissues(hypothalamus), in kidney, heart and spleen. In addition, prosaposinshows high expression in brown adipose tissue (BAT) and white adiposetissue (WAT) (see FIG. 4A). Brown adipose tissue is a well-characterizedtissue which is well developed in newborn mammals, including humans. Oneimportant task of BAT is to generate heat and maintain body temperaturehomeostasis in newborn. Thus, an expression of the protein of theinvention in adipose tissues is confirming a role in the regulation ofenergy homeostasis and thermogenesis.

Further, we show that mammalian prosaposin is regulated by fasting andby genetically induced obesity. In this invention, we used mouse modelsof insulin resistance and/or diabetes, such as mice carrying geneknockouts in the leptin pathway (for example, ob (leptin) or db (leptinreceptor) mice) to study the expression of prosaposin. Such mice developtypical symptoms of diabetes, show hepatic lipid accumulation andfrequently have increased plasma lipid levels (see Bruning et al, 1998,Mol. Cell. 2: 559-569). We found, for example, that the expression ofprosaposin is strongly upregulated in liver of fasted and ob/ob mice(see FIG. 4B). In addition, a marked upregulation can be observed in themetabolically active tissue (for example, white adipose tissue (WAT)) ofgenetically obese (ob/ob) (see FIG. 4B).

Susceptible wild type mice (for example C57BI/6) show symptoms ofdiabetes lipid accumulation, and high plasma lipid levels, if fed a highfat diet. In such mice, the most prominent response with regard tometabolically active tissues was observed. In those mice, the expressionof prosaposin is significantly enhanced in white adipose tissue (seeFIG. 4C) and in liver and muscle tissues, supporting a hypothesis thatthe protein of the invention is a modulator of adipogenesis.

In addition, we show in this invention that the mRNA of the protein ofthe invention is up-regulated during adipocyte differentiation in vitro(see EXAMPLES for more detail), suggesting a role as modulator ofadipocyte lipid accumulation. With regard to changes in expressionintensity during the differentiation of preadipocytes to adipocytes, anenhancement in relative signal intensity can be observed for the proteinof the invention during the in vitro differentiation program of 3T3-L1(see FIG. 4D). Thus, we conclude that prosaposin or variants orprocessing products thereof have a function in the metabolism of matureadipocytes.

Microarrays are analytical tools routinely used in bioanalysis. Amicroarray has molecules distributed over, and stably associated with,the surface of a solid support. The term “microarray” refers to anarrangement of a plurality of polynucleotides, polypeptides, antibodies,or other chemical compounds on a substrate. Microarrays of polypeptides,polynucleotides, and/or antibodies have been developed and find use in avariety of applications, such as monitoring gene expression, drugdiscovery, gene sequencing, gene mapping, bacterial identification, andcombinatorial chemistry. One area in particular in which microarraysfind use is in gene expression analysis (see Example 4). Arraytechnology can be used to explore the expression of a single polymorphicgene or the expression profile of a large number of related or unrelatedgenes. When the expression of a single gene is examined, arrays areemployed to detect the expression of a specific gene or its variants.When an expression profile is examined, arrays provide a platform foridentifying genes that are tissue specific, are affected by a substancebeing tested in a toxicology assay, are part of a signaling cascade,carry out housekeeping functions, or are specifically related to aparticular genetic predisposition, condition, disease, or disorder.

Microarrays may be prepared, used, and analyzed using methods known inthe art (see for example, Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796, Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/251116;Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R. A. etal. (1997) Proc. Natl. Acad. Sci. USA 94:21502155; Heller, M. J. et al.(1997) U.S. Pat. No. 5,605,662). Various types of microarrays are wellknown and thoroughly described in Schena, M., ed. (1999; DNAMicroarrays: A Practical Approach, Oxford University Press, London).

Oligonucleotides or longer fragments derived from any of thepolynucleotides described herein may be used as elements on amicroarray. The microarray can be used in transcript imaging techniqueswhich monitor the relative expression levels of large numbers of genessimultaneously as described below. The microarray may also be used toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, to monitorprogression/regression of disease as a function of gene expression, andto develop and monitor the activities of therapeutic agents in thetreatment of disease. In particular, this information may be used todevelop a pharmacogenomic profile of a patient in order to select themost appropriate and effective treatment regimen for that patient. Forexample, therapeutic agents which are highly effective and display thefewest side effects may be selected for a patient based on his/herpharmacogenomic profile.

As determined by microarray analysis, prosaposin shows differentialexpression in human primary adipocytes (see FIG. 5A) and a humanadipocyte cell line (see FIG. 5B). The expression of prosaposin isstrongly upregulated during the human adipocyte differentiation (seeFIG. 5). Thus, the prosaposin protein in preadipocyctes issignifficantly enhancing adipose differentiation at a very early stage.Therefore, prosaposin plays an essential role in adipogenesis. Inaddition, the results strongly suggest a role of prosaposin in theregulation in human metabolism, for example, as modulator (for example,enhancer) of adipogenesis. Thus, prosaposin is a strong candidate for apharmaceutical composition for the treatment of conditions related tohuman metabolism, such as obesity, diabetes, and/or metabolic syndrome.

This invention further shows that prosaposin induces the differentiationof insulin-producing cells and is thus a target for the treatment ofdiabetes. In connection with the present invention, the term “progenitorcells” relates to undifferentiated cells capable of being differentiatedinto insulin producing cells. The term particularly includes stem cells,i.e. undifferentiated or immature embryonic, adult, or somatic cellsthat can give rise to various specialized cell types. The term “stemcells” can include embryonic stem cells (ES) and primordial germ cells(EG) cells of mammalian, e.g. human or animal origin. Isolation andculture of such cells is well known to those skilled in the art (see,for example, Thomson et al., (1998) Science 282: 1145-1147; Shamblott etal., (1998) Proc. Natl. Acad. Sci. USA 95: 13726-13731; U.S. Pat. No.6,090,622; U.S. Pat. No. 5,914,268; WO 00/27995; Notarianni et al.,(1990) J. Reprod. Fert. 41: 51-56; Vassilieva et al., (2000) Exp. Cell.Res. 258: 361-373). Adult or somatic stem cells have been identified innumerous different tissues such as intestine, muscle, bone marrow,liver, and brain. WO 03/023018 describes a novel method for isolating,culturing, and differentiating intestinal stem cells for therapeuticuse. In the pancreas, several indications suggest that stem cells arealso present within the adult tissue (Gu and Sarvetnick, (1993)Development 118: 33-46; Bouwens, (1998) Microsc Res Tech 43: 332-336;Bonner-Weir, (2000) J. Mol. Endocr. 24: 297-302).

Embryonic stem cells can be isolated from the inner cell mass ofpre-implantation embryos (ES cells) or from the primordial germ cellsfound in the genital ridges of post-implanted embryos (EG cells). Whengrown in special culture conditions such as spinner culture or hangingdrops, both ES and EG cells aggregate to form embryoid bodies (EB). EBsare composed of various cell types similar to those present duringembryogenesis. When cultured in appropriate media, EB can be used togenerate in vitro differentiated phenotypes, such as extraembryonicendoderm, hematopoietic cells, neurons, cardiomyocytes, skeletal musclecells, and vascular cells. We have previously described a method thatallows EB to efficiently differentiate into insulin-producing cells (asdescribed in patent application PCT/EP02/04362, published as WO02/086107 and by Blyszczuk et al., (2003) Proc Natl Acad Sci USA 100:998-1003, which are incorporated herein by reference).

The results shown in FIG. 6 clearly demonstrate an induction of thedifferentiation of insulin-producing and glucose-responsive cells byprosaposin. Thus, prosaposin can induce the differentiation ofbeta-cells and is therefore a target for therapeutic uses in thetreatment of diabetes, for example, when regeneration of cells isrequired.

Before the present invention is described, it is understood that alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

In the present invention the term “beta-cell regeneration” refers to anat least partial restoration of normal beta-cell function by increasingthe number of functional insulin secreting beta-cells and/or byrestoring normal function in functionally impaired beta-cells.

The invention also encompasses polynucleotides that encode the proteinsof the invention and homologous proteins. Accordingly, any nucleic acidsequence, which encodes the amino acid sequences of the proteins of theinvention and homologous proteins, can be used to generate recombinantmolecules that express the proteins of the invention and homologousproteins. In a particular embodiment, the invention encompasses anucleic acid encoding Drosophila saposin-related, or their mammalian,e.g. human homologs; referred to herein as the proteins of theinvention. It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding the proteins, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. The invention contemplates each and every possible variationof nucleotide sequence that can be made by selecting combinations basedon possible codon choices.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those of the polynucleotide encoding the proteins of theinvention, under various conditions of stringency. Hybridizationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex or probe, as taught in Wahl & Berger (1987: MethodsEnzymol. 152: 399-407) and Kimmel (1987; Methods Enzymol. 152: 507-511),and may be used at a defined stringency. Preferably, hybridization understringent conditions means that after washing for 1 h with 1×SSC and0.1% SDS at 50° C., preferably at 55° C., more preferably at 62° C. andmost preferably at 65° C., particularly for 1 h in 0.2×SSC and 0.1% SDSat 50° C., preferably at 55° C., more preferably at 62° C. and mostpreferably at 65° C., a positive hybridization signal is observed.Altered nucleic acid sequences encoding the proteins which areencompassed by the invention include deletions, insertions orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent protein.

The encoded proteins may also contain deletions, insertions orsubstitutions of amino acid residues, which produce a silent change andresult in functionally equivalent proteins. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the biological activity ofthe protein is retained. Furthermore, the invention relates to peptidefragments of the proteins or derivatives thereof such as cyclicpeptides, retro-inverso peptides or peptide mimetics having a length ofat least 4, preferably at least 6 and up to 50 amino acids.

Also included within the scope of the present invention are alleles ofthe genes encoding the proteins of the invention and homologousproteins. As used herein, an ‘allele’ or ‘allelic sequence’ is analternative form of the gene, which may result from at least onemutation in the nucleic acid sequence. Alleles may result in alteredmRNAs or polypeptides whose structures or function may or may not bealtered. Any given gene may have none, one or many allelic forms. Commonmutational changes, which give rise to alleles, are generally ascribedto natural deletions, additions or substitutions of nucleotides. Each ofthese types of changes may occur alone or in combination with theothers, one or more times in a given sequence.

The nucleic acid sequences encoding the proteins of the invention andhomologous proteins may be extended utilizing a partial nucleotidesequence and employing various methods known in the art to detectupstream sequences such as promoters and regulatory elements.

In order to express a biologically active protein, the nucleotidesequences encoding the proteins or functional equivalents, may beinserted into appropriate expression vectors, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods, which are well known to thoseskilled in the art, may be used to construct expression vectorscontaining sequences encoding the proteins and the appropriatetranscriptional and translational control elements. Regulatory elementsinclude for example a promoter, an initiation codon, a stop codon, amRNA stability regulatory element, and a polyadenylation signal.Expression of a polynucleotide can be assured by (i) constitutivepromoters such as the Cytomegalovirus (CMV) promoter/enhancer region,(ii) tissue specific promoters such as the insulin promoter (see, Soriaet al., 2000, Diabetes 49: 157-162), SOX2 gene promotor (see Li et al.,(1998) Curr. Biol. 8: 971-974), Msi-1 promotor (see Sakakibara et al.,(1997) J. Neuroscience 17: 8300-8312), alpha-cardia myosin heavy chainpromotor or human atrial natriuretic factor promotor (Klug et al.,(1996) J. Clin. Invest 98: 216-224; Wu et al., (1989) J. Biol. Chem.264: 6472-6479) or (iii) inducible promoters such as the tetracyclineinducible system. Expression vectors can also contain a selection agentor marker gene that confers antibiotic resistance such as the neomycin,hygromycin or puromycin resistance genes. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook, J. et al.(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y. and Ausubel, F. M. et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y.

In a further embodiment of the invention, natural, modified orrecombinant nucleic acid sequences encoding the proteins of theinvention and homologous proteins may be ligated to a heterologoussequence to encode a fusion protein.

A variety of expression vector/host systems may be utilized to containand express sequences encoding the proteins or fusion proteins. Theseinclude, but are not limited to, micro-organisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus, adenovirus, adeno-associated virus, lentiverus,retrovirus); plant cell systems transformed with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or with bacterial expression vectors (e.g., Ti or PBR322 plasmids);or animal cell systems.

The presence of polynucleotide sequences of the invention in a samplecan be detected by DNA-DNA or DNA-RNA hybridization and/or amplificationusing probes or portions or fragments of said polynucleotides. Nucleicacid amplification based assays involve the use of oligonucleotides oroligomers based on the sequences specific for the gene to detecttransformants containing DNA or RNA encoding the corresponding protein.As used herein ‘oligonucleotides’ or ‘oligomers’ refer to a nucleic acidsequence of at least about 10 nucleotides and as many as about 60nucleotides, preferably about 15 to 30 nucleotides, and more preferablyabout 20-25 nucleotides, which can be used as a probe or amplimer.

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting polynucleotide sequences include oligo-labeling, nicktranslation, end-labeling of RNA probes, PCR amplification using alabeled nucleotide, or enzymatic synthesis. These procedures may beconducted using a variety of commercially available kits (Pharmacia &Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. BiochemicalCorp., (Cleveland, Ohio).

The presence of proteins of the invention in a sample can be determinedby immunological methods or activity measurement. A variety of protocolsfor detecting and measuring the expression of proteins, using eitherpolyclonal or monoclonal antibodies specific for the protein or reagentsfor determining protein activity are known in the art. Examples includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on the protein is preferred, but a competitivebinding assay may be employed. These and other assays are described,among other places, in Hampton, R. et al. (1990; Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al.(1983; J. Exp. Med. 158: 1211-1216).

Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent or chromogenicagents as well as substrates, co-factors, inhibitors, magneticparticles, and the like.

Host cells transformed with nucleotide sequences encoding a protein ofthe invention may be cultured under conditions suitable for theexpression and recovery of said protein from cell culture. The proteinproduced by a recombinant cell may be secreted or containedintracellularly depending on the sequence or/and the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining polynucleotides, which encode the protein may be designed tocontain signal sequences, which direct secretion of the protein througha prokaryotic or eukaryotic cell membrane. Other recombinantconstructions may be used to join sequences encoding the protein tonucleotide sequence encoding a polypeptide domain, which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAG extension/affinitypurification system (Immunex Corp., Seattle, Wash.) The inclusion ofcleavable linker sequences such as those specific for Factor XA orEnterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and the desired protein may be used to facilitate purification.

Diagnostics and Therapeutics

The data disclosed in this invention show that the nucleic acids andproteins of the invention and effector/modulator molecules thereof areuseful in diagnostic and therapeutic applications implicated, forexample, but not limited to, metabolic syndrome including obesity,diabetes mellitus, eating disorder, cachexia, hypertension, coronaryheart disease, hypercholesterolemia (dyslipidemia), and/or gallstones.Hence, diagnostic and therapeutic uses for the proteins of the inventionnucleic acids and proteins of the invention are, for example but notlimited to, the following: (i) tissue regeneration in vitro and in vivo(regeneration for all these tissues and cell types composing thesetissues and cell types derived from these tissues), (ii) small moleculedrug target, (iii) antibody target (therapeutic, diagnostic, drugtargeting/cytotoxic antibody), (iv) diagnostic and/or prognostic marker,(v) protein therapy, (vi) gene therapy (gene delivery/gene ablation),and (vii) research tools.

According to this invention the prosaposin product may be administered

-   i) as a pharmaceutical composition e.g. enterally, parenterally or    topically, preferably directly to the pancreas,-   ii) via implantation of prosaposin protein product expressing cells,    and/or-   iii) via gene therapy    as described in more detail below.

Further, the prosaposin expression level in a patient might beinfluenced by a prosaposin modulator/effector administered

-   i) as a pharmaceutical composition e.g. enterally, parenterally or    topically, preferably directly to the pancreas,-   ii) via cell based therapy, and/or-   iii) via gene therapy    as described in more detail below.

The prosaposin product or the prosaposin modulator/effector, i.e. apharmaceutically active substance influencing, particularly increasingthe prosaposin expression level or function may be administered in theabove described manner alone or in combination with anotherpharmaceutical composition useful to treat beta-cell degeneration, forexample hormones, growth factors or immune modulating agents.

A prosaposin product or a modulator/effector thereof may be administeredin patients suffering from a disease going along with impaired beta-cellfunction, for example but not limited to diabetes type I, LADA, orprogressed diabetes type II. It is further contemplated that aprosaposin product or the modulator/effector thereof may be administeredpreventively to patients at risk to develop beta-cell degeneration, likefor example but not limited to patients suffering from diabetes type IIor LADA in early stages. A variety of pharmaceutical formulations anddifferent delivery techniques are described in further detail below.

The present invention also relates to methods for differentiatingprogenitor cells into insulin-producing cells in vitro comprising

-   (a) activating one or more pancreatic genes in a progenitor, e.g.    stem cell (optional step, particularly if embryonic stem cells are    used)-   (b) aggregating said cells to form embryoid bodies (optional step,    particularly if embryonic stem cells are used)-   (c) cultivating embryoid bodies or cultivating adult stem cells    (e.g., duct cells) in specific differentiation media containing a    prosaposin protein product and/or a modulator/effector thereof under    conditions wherein beta-cell differentiation is significantly    enhanced, and-   (d) identifying and selecting insulin-producing cells.

Activation of pancreatic genes may comprise transfection of a cell withpancreatic gene operatively linked to an expression control sequence,e.g. on a suitable transfection vector, as described in WO 03/023018,which is herein incorporated by reference. Examples of preferredpancreatic genes are Pdx1, Pax4, Pax6, neurogenin 3 (ngn3), Nkx 6.1, Nkx6.2, Nkx 2.2, HB 9, BETA2/Neuro D, Isl 1, HNF1-alpha, HNF1-beta and HNF3of human or animal origin. Each gene can be used individually or incombination with at least one other gene. Pax4 is especially preferred.

Prosaposin products, e.g. prosaposin protein or nucleic acid products,are preferably produced via recombinant techniques because such methodsare capable of achieving high amounts of protein at a great purity, butare not limited to products expressed in bacterial, plant, mammalian, orinsect cell systems.

Further, the data show that the prosaposin nucleic acids and proteinsand effector/modulator molecules thereof are useful for the modulation,e.g. stimulation, of pancreatic development and/or for the regenerationof pancreatic cells or tissues, e.g. cells having exocrinous functionssuch as acinar cells, centroacinar cells and/or ductal cells, and/orcells having endocrinous functions, particularly cells in Langerhansislets such as alpha-, beta-, delta- and/or PP-cells, more particularlybeta-cells.

The nucleic acids and proteins of the invention and effectors thereofare useful in diagnostic and therapeutic applications implicated invarious applications as described below. For example, but not limitedto, cDNAs encoding the proteins of the invention and particularly theirhuman homologues may be useful in gene therapy, and the proteins of theinvention and particularly their human homologues may be useful whenadministered to a subject in need thereof. By way of non-limitingexample, the compositions of the present invention will have efficacyfor treatment of patients suffering from, for example, but not limitedto, in metabolic disorders as described above.

Prosaposin product cell therapy, i.e. pancreatic implantation of cellsproducing prosaposin protein product, is also contemplated. Thisembodiment would involve implanting cells capable of synthesizing andsecreting a biologically active form of prosaposin protein product intopatients. Such prosaposin protein product-producing cells may be cellsthat are natural producers of prosaposin protein product or may be cellsthat are modified to express the protein. Such modified cells includerecombinant cells whose ability to produce a prosaposin protein producthas been augmented by transformation with a gene encoding the desiredprosaposin protein product in a vector suitable for promoting itsexpression and secretion. In order to minimize a potential immunologicalreaction in patients being administered prosaposin protein product of aforeign species, it is preferred that the cells producing prosaposinprotein product be of human origin and produce human prosaposin proteinproduct. Likewise, it is preferred that the recombinant cells producingprosaposin protein product be transformed with an expression vectorcontaining a gene encoding a human prosaposin protein product. Implantedcells may be encapsulated to avoid infiltration of surrounding tissue.Human or nonhuman animal cells may be implanted in patients inbiocompatible, semipermeable polymeric enclosures or membranes thatallow release of prosaposin protein product, but that preventdestruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissue.

Alternatively, prosaposin protein product secreting cells may beintroduced into a patient in need intraportally via a percutaneoustranshepatic approach using local anaesthesia. Between 3000 and 100 000equivalent differentiated insulin-producing cells per kilogram bodyweight are preferably administered. Such surgical techniques are wellknown in the art and can be applied without any undue experimentation,see Pyzdrowski et al, 1992, New England J. Medicine 327:220-226; Heringet al., Transplantation Proc. 26:570-571, 1993; Shapiro et al., NewEngland J. Medicine 343:230-238, 2000.

In a further preferred embodiment, prosaposin protein product can bedelivered directly to progenitor, e.g. stem cells in order to stimulatethe differentiation of insulin producing cells. For example, proteindelivery can be achieved by polycationic liposomes (Sells et al. (1995)Biotechniques 19:72-76), Tat-mediated protein transduction (Fawell etal. (1993) Proc. Natl. Acad. Sci. USA 91:664-668) and by fusing aprotein to the cell permeable motif derived from the PreS2-domain of thehepatitis-B virus (Oess and Hildt (2000) Gene Ther. 7:750-758).Preparation, production and purification of such proteins from bacteria,yeast or eukaryotic cells are well known by persons skilled in the art.In this embodiment of the invention, prosaposin may be added preferablyat concentrations between 1 ng/ml and 500 ng/ml, more preferably between10 and 100 ng/ml, e.g. at about 50 ng/ml.

Further, the invention relates to a cell preparation comprisingdifferentiated progenitor cells, e.g. stem cells exhibiting insulinproduction, particularly an insulin-producing cell line obtainable bythe method described above. The insulin-producing cells may exhibit astable or a transient expression of at least one pancreatic geneinvolved in beta-cell differentiation. The cells are preferably humancells that are derived from human stem cells. For therapeuticapplications the production of autologous human cells from adult stemcells of a patient is especially preferred. However, the insulinproducing cells may also be derived from non-autologous cells. Ifnecessary, undesired immune reactions may be avoided by encapsulation,immunosuppression and/or modulation or due to non-immunogenic propertiesof the cells.

The insulin producing cells of the invention preferably exhibitcharacteristics that closely resemble naturally occurring beta-cells.Further, the cells of the invention preferably are capable of a quickresponse to glucose. After addition of 27.7 mM glucose, the insulinproduction is enhanced by a factor of at least 2, preferably by a factorof at least 3. Further, the cells of the invention are capable ofnormalizing blood glucose levels after transplantation into mice.

The invention further encompasses functional pancreatic cells obtainableor obtained by the method according to the invention. The cells arepreferably of mammalian, e.g. human origin. Preferably, said cells arepancreatic beta-cells, e.g. mature pancreatic beta-cells or stem cellsdifferentiated into pancreatic beta-cells. Such pancreatic beta cellspreferably secrets insulin in response to glucose. Moreover, the presentinvention provides functional pancreatic cell that express glucagon inresponse to glucose. A preparation comprising the cells of the inventionmay additionally contain cells with properties of other endocrine celltypes such as alpha-cells, delta-cells and/or PP-cells. These cells arepreferably human cells.

The cell preparation of the invention is preferably a pharmaceuticalcomposition comprising the cells together with pharmacologicallyacceptable carriers, diluents and/or adjuvants. The pharmaceuticalcomposition is preferably used for the treatment or prevention ofpancreatic diseases, e.g. diabetes.

According to the present invention, the functional insulin producingcells treated with prosaposin may be transplanted preferablyintrahepatic, directly into the pancreas of an individual in need, or byother methods. Alternatively, such cells may be enclosed intoimplantable capsules that can be introduced into the body of anindividual, at any location, more preferably in the vicinity of thepancreas, or the bladder, or the liver, or under the skin. Methods ofintroducing cells into individuals are well known to those of skill inthe art and include, but are not limited to, injection, intravenous orparenteral administration. Single, multiple, continuous or intermittentadministration can be effected. The cells can be introduced into any ofseveral different sites, including but not limited to the pancreas, theabdominal cavity, the kidney, the liver, the celiac artery, the portalvein or the spleen. The cells may also be deposited in the pancreas ofthe individual.

The methodology for the membrane encapsulation of living cells isfamiliar to those of ordinary skill in the art, and the preparation ofthe encapsulated cells and their implantation in patients may beaccomplished without undue experimentation. See, e.g., U.S. Pat. Nos.4,892,538, 5,011,472, and 5,106,627, each of which is specificallyincorporated herein by reference. A system for encapsulating livingcells is described in PCT Application WO 91/10425 of Aebischer et al.,specifically incorporated herein by reference. See also, PCP ApplicationWO 91/10470 of Aebischer et al., Winn et al., Exper. Neurol., 113:322-329, 1991, Aebischer et al., Exper. Neurol., 11 1:269-275, 1991;Tresco et al., ASAIO, 38:17-23, 1992, each of which is specificallyincorporated herein by reference. Techniques for formulating a varietyof other sustained- or controlled-delivery means, such as liposomecarriers, bio-erodible particles or beads and depot injections, are alsoknown to those skilled in the art.

In another embodiment gene therapy ex vivo is envisioned, i.e. thepatient's own cells may be transformed ex vivo to produce a prosaposinprotein product or a protein stimulating prosaposin expression and wouldbe directly reimplanted. For example, cells retrieved from the patientmay be cultured and transformed with an appropriate vector. After anoptional propagation/expansion phase, the cells can be transplanted backinto the same patient's body, particularly the pancreas, where theywould produce and release the desired prosaposin protein product.Delivery by transfection and by liposome injections may be achievedusing methods, which are well known in the art. Any of the therapeuticmethods described above may be applied to any suitable subjectincluding, for example, mammals such as dogs, cats, cows, horses,rabbits, monkeys, and most preferably, humans.

Prosaposin product gene therapy in vivo is also envisioned, byintroducing the gene coding for a prosaposin protein product intotargeted pancreas cells via local injection of a nucleic acid constructor other appropriate delivery methods (Hefti, J. Neurobiol.,25:1418-1435, 1994). For example, a nucleic acid sequence encoding aprosaposin protein product may be contained in an adeno-associated virusvector or adenovirus vector for delivery to the pancreas cells.Alternative viral vectors include, but are not limited to, retrovirus,herpes simplex virus and papilloma virus vectors. Physical transfer,either in vivo or ex vivo as appropriate, may also be achieved byliposome-mediated transfer, direct injection (naked DNA),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation or microparticle bombardment (gene gun).

Immunosuppressive drugs, such as cyclosporin, can also be administeredto the patient in need to reduce the host reaction versus graft.Allografts using the cells obtained by the methods of the presentinvention are also useful because a single healthy donor could supplyenough cells to regenerate at least partial pancreas function inmultiple recipients.

Administration of a prosaposin protein product and/ormodulators/effectors thereof in a pharmaceutical composition to asubject in need thereof, particularly a human patient, leads to an atleast partial regeneration of pancreatic cells. Preferably, these cellsare insulin producing beta-cells that will contribute to the improvementof a diabetic state. With the administration of this composition e.g. ona short term or regular basis, an increase in beta-cell mass can beachieved. This effect upon the body reverses the condition of diabetespartially or completely. As the subject's blood glucose homeostasisimproves, the dosage administered may be reduced in strength. In atleast some cases further administration can be discontinued entirely andthe subject continues to produce a normal amount of insulin withoutfurther treatment. The subject is thereby not only treated but could becured entirely of a diabetic condition. However, even moderateimprovements in beta-cell mass can lead to a reduced requirement forexogenous insulin, improved glycemic control and a subsequent reductionin diabetic complications. In another example, the compositions of thepresent invention will also have efficacy for treatment of patients withother pancreatic diseases such as pancreatic cancer, dysplasia, orpancreatitis, if beta-cells are to be regenerated.

The nucleic acids of the invention or fragments thereof, may further beuseful in diagnostic applications, wherein the presence or amount of thenucleic acids or the proteins are to be assessed. Further antibodiesthat bind immunospecifically to the novel substances of the inventionmay be used in therapeutic or diagnostic methods.

For example, in one aspect, antibodies, which are specific for theproteins of the invention and homologous proteins, may be used directlyas an effector, e.g. an antagonist or indirectly as a targeting ordelivery mechanism for bringing a pharmaceutical agent to cells ortissue which express the protein. The antibodies may be generated usingmethods that are well known in the art. Such antibodies may include, butare not limited to, polyclonal, monoclonal, chimeric single chain, Fabfragments, and fragments produced by a Fab expression library.Neutralising antibodies, (i.e., those which inhibit dimer formation) areespecially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith the protein or any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. It is preferred that thepeptides, fragments or oligopeptides used to induce antibodies to theprotein have an amino acid sequence consisting of at least five aminoacids, and more preferably at least 10 amino acids.

Monoclonal antibodies to the proteins may be prepared using anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Köhler, G. et al. (1975) Nature 256: 495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81: 31-42; Cote, R. J. etal. Proc. Natl. Acad. Sci. 80: 2026-2030; Cole, S. P. et al. (1984) Mol.Cell Biol. 62: 109-120).

In addition, techniques developed for the production of ‘chimericantibodies’, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81: 6851-6855; Neuberger, M. S. et al (1984) Nature312: 604-608; Takeda, S. et al. (1985) Nature 314: 452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to producesingle chain antibodies specific for the proteins of the invention andhomologous proteins. Antibodies with related specificity, but ofdistinct idiotypic composition, may be generated by chain shuffling fromrandom combinatorial immunoglobulin libraries (Burton, D. R. (1991)Proc. Natl. Acad. Sci. 88: 11120-11123). Antibodies may also be producedby inducing in vivo production in the lymphocyte population or byscreening recombinant immunoglobulin libraries or panels of highlyspecific binding reagents as disclosed in the literature (Orlandi, R. etal. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al.(1991) Nature 349: 293-299).

Antibody fragments, which contain specific binding sites for theproteins may also be generated. For example, such fragments include, butare not limited to, the F(ab′)₂ fragments which can be produced byPepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254: 1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding and immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between the protein and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reacive totwo non-interfering protein epitopes are preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides or fragmentsthereof or nucleic acid effector molecules such as antisense molecules,aptamers, RNAi molecules or ribozymes may be used for therapeuticpurposes. In one aspect, aptamers, i.e. nucleic acid molecules, whichare capable of binding to a protein of the invention and modulating itsactivity, may be generated by a screening and selection procedureinvolving the use of combinatorial nucleic acid libraries.

In a further aspect, antisense molecules may be used in situations inwhich it would be desirable to block the transcription of the mRNA Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding the proteins of the invention and homologousproteins. Thus, antisense molecules may be used to modulate proteinactivity or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding the proteins. Expression vectorsderived from retroviruses, adenovirus, herpes or vaccinia viruses orfrom various bacterial plasmids may be used for delivery of nucleotidesequences to the targeted organ, tissue or cell population. Methods,which are well known to those skilled in the art, can be used toconstruct recombinant vectors, which will express antisense moleculescomplementary to the polynucleotides of the genes encoding the proteinsof the invention and homologous proteins. These techniques are describedboth in Sambrook et al. (supra) and in Ausubel et al. (supra). Genesencoding the proteins of the invention and homologous proteins can beturned off by transforming a cell or tissue with expression vectors,which express high levels of polynucleotides that encode the proteins ofthe invention and homologous proteins or fragments thereof. Suchconstructs may be used to introduce untranslatable sense or antisensesequences into a cell. Even in the absence of integration into the DNA,such vectors may continue to transcribe RNA molecules until they aredisabled by endogenous nucleases. Transient expression may last for amonth or more with a non-replicating vector and even longer ifappropriate replication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, e.g. DNA, RNA or nucleic acid analoguessuch as PNA, to the control regions of the genes encoding the proteinsof the invention and homologous proteins, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit cause inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In; Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage.Examples, which may be used, include engineered hammerhead motifribozyme molecules that can be specifically and efficiently catalyzeendonucleolytic cleavage of sequences encoding the proteins of theinvention and homologous proteins. Specific ribozyme cleavage siteswithin any potential RNA target are initially identified by scanning thetarget molecule for ribozyme cleavage sites which include the followingsequences: GUA, GUU, and GUC. Once identified, short RNA sequences ofbetween 15 and 20 ribonucleotides corresponding to the region of thetarget gene containing the cleavage site may be evaluated for secondarystructural features which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Nucleic acid effector/modulator molecules, e.g. antisense molecules andribozymes of the invention may be prepared by any method known in theart for the synthesis of nucleic acid molecules. These includetechniques for chemically synthesizing oligonucleotides such as solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in Vivo transcription of DNA sequences.Such DNA sequences may be incorporated into a variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize antisense RNA constitutively orinducibly can be introduced into cell lines, cells or tissues. RNAmolecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculeor modifications in the nucleobase, sugar and/or phosphate moieties,e.g. the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of non-traditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods, which are well known in the art. Any of thetherapeutic methods described above may be applied to any suitablesubject including, for example, mammals such as dogs, cats, cows,horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of the nucleic acids andthe proteins of the invention and homologous nucleic acids or proteins,antibodies to the proteins of the invention and homologous proteins,mimetics, agonists, antagonists or inhibitors of the proteins of theinvention and homologous proteins or nucleic acids. The compositions maybe administered alone or in combination with at least one other agent,such as stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone or in combination with other agents,drugs or hormones. The pharmaceutical compositions utilized in thisinvention may be administered by any number of routes including, but notlimited to, oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, intraventricular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingualor rectal means.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of the activecompounds into preparations, which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart. For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of preadipocytecell lines or in animal models, usually mice, rabbits, dogs or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans. Atherapeutically effective dose refers to that amount of activeingredient, for example the nucleic acids or the proteins of theinvention or fragments thereof or antibodies, which is sufficient fortreating a specific condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions, which exhibit large therapeutic indices,are preferred. The data obtained from cell culture assays and animalstudies is used in formulating a range of dosage for human use. Thedosage contained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosagefrom employed, sensitivity of the patient, and the route ofadministration. The exact dosage will be determined by the practitioner,in light of factors related to the subject that requires treatment.Dosage and administration are adjusted to provide sufficient levels ofthe active moiety or to maintain the desired effect. Factors, which maybe taken into account, include the severity of the disease state,general health of the subject, age, weight, and gender of the subject,diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek or once every two weeks depending on half-life and clearance rateof the particular formulation. Normal dosage amounts may vary from 0.1to 100,000 g, up to a total dose of about 1 g, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

In another embodiment, antibodies which specifically bind to theproteins may be used for the diagnosis of conditions or diseasescharacterized by or associated with over- or underexpression of theproteins of the invention and homologous proteins or in assays tomonitor patients being treated with the proteins of the invention andhomologous proteins, or effectors thereof, e.g. agonists, antagonists,or inhibitors. Diagnostic assays include methods which utilize theantibody and a label to detect the protein in human body fluids orextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by joining them, either covalently ornon-covalently, with a reporter molecule. A wide variety of reportermolecules, which are known in the art may be used several of which aredescribed above.

A variety of protocols including ELISA, RIA, and FACS for measuringproteins are known in the art and provide a basis for diagnosing alteredor abnormal levels of gene expression. Normal or standard values forgene expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibodies to the protein under conditions suitable for complexformation. The amount of standard complex formation may be quantified byvarious methods, but preferably by photometric means. Quantities ofprotein expressed in control and disease, samples e.g. from biopsiedtissues are compared with the standard values. Deviation betweenstandard and subject values establishes the parameters for diagnosingdisease.

In another embodiment of the invention, the polynucleotides specific forthe proteins of the invention and homologous proteins may be used fordiagnostic purposes. The polynucleotides, which may be used, includeoligonucleotide sequences, antisense RNA and DNA molecules, and PNAs.The polynucleotides may be used to detect and quantitate gene expressionin biopsied tissues in which gene expression may be correlated withdisease. The diagnostic assay may be used to distinguish betweenabsence, presence, and excess gene expression, and to monitor regulationof protein levels during therapeutic intervention.

In one aspect, hybridization with probes which are capable of detectingpolynucleotide sequences, including genomic sequences, encoding theproteins of the invention and homologous proteins or closely relatedmolecules, may be used to identify nucleic acid sequences which encodethe respective protein. The hybridization probes of the subjectinvention may be DNA or RNA and are preferably derived from thenucleotide sequence of the polynucleotide encoding the proteins of theinvention or from a genomic sequence including promoter, enhancerelements, and introns of the naturally occurring gene. Hybridizationprobes may be labeled by a variety of reporter groups, for example,radionuclides such as ³²P or ³⁵S or enzymatic labels, such as alkalinephosphatase coupled to the probe via avidin/biotin coupling systems, andthe like.

Polynucleotide sequences specific for the proteins of the invention andhomologous nucleic acids may be used for the diagnosis of conditions ordiseases, which are associated with the expression of the proteins.Examples of such conditions or diseases include, but are not limited to,metabolic diseases and disorders, including obesity, diabetes, and/ormetabolic syndrome. Polynucleotide sequences specific for the proteinsof the invention and homologous proteins may also be used to monitor theprogress of patients receiving treatment for metabolic diseases anddisorders, including obesity, diabetes, and/or metabolic syndrome. Thepolynucleotide sequences may be used qualitative or quantitative assays,e.g. in Southern or Northern analysis, dot blot or other membrane-basedtechnologies; in PCR technologies; or in dip stick, pin, ELISA or chipassays utilizing fluids or tissues from patient biopsies to detectaltered gene expression.

In a particular aspect, the nucleotide sequences specific for theproteins of the invention and homologous nucleic acids may be useful inassays that detect activation or induction of various metabolic diseasesand disorders, including obesity, diabetes, and/or metabolic syndrome.The nucleotide sequences may be labeled by standard methods, and addedto a fluid or tissue sample from a patient under conditions suitable forthe formation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. The presence of altered levels of nucleotidesequences encoding the proteins of the invention and homologous proteinsin the sample indicates the presence of the associated disease. Suchassays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies, in clinical trials orin monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of a disease associatedwith expression of the proteins of the invention and homologousproteins, a normal or standard profile for expression is established.This may be accomplished by combining body fluids or cell extracts takenfrom normal subjects, either animal or human, with a sequence or afragment thereof, which is specific for the nucleic acids encoding theproteins of the invention and homologous nucleic acids, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease. Once disease isestablished and a treatment protocol is initiated, hybridization assaysmay be repeated on a regular basis to evaluate whether the level ofexpression in the patient begins to approximate that, which is observedin the normal patient. The results obtained from successive assays maybe used to show the efficacy of treatment over a period ranging fromseveral days to months.

With respect to metabolic diseases such as described above the presenceof an unusual amount of transcript in biopsied tissue from an individualmay indicate a predisposition for the development of the disease or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the metabolic diseases and disorders.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding the proteins of the invention and homologous proteinsmay involve the use of PCR. Such oligomers may be chemicallysynthesized, generated enzymatically or produced from a recombinantsource. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5prime.fvdarw.3prime) and another withantisense (3prime.rarw.5prime), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantification of closely related DNA or RNA sequences.

In another embodiment of the invention, the nucleic acid sequences mayalso be used to generate hybridization probes, which are useful formapping the naturally occurring genomic sequence. The sequences may bemapped to a particular chromosome or to a specific region of thechromosome using well known techniques. Such techniques include FISH,FACS or artificial chromosome constructions, such as yeast artificialchromosomes, bacterial artificial chromosomes, bacterial P1constructions or single chromosome cDNA libraries as reviewed in Price,C. M. (1993) Blood Rev. 7: 127-134, and Trask, B. J. (1991) TrendsGenet. 7:149-154. FISH (as described in Verma et al. (1988) HumanChromosomes: A Manual of Basic Techniques, Pergamon Press, New York,N.Y.). The results may be correlated with other physical chromosomemapping techniques and genetic map data. Examples of genetic map datacan be found in the 1994 Genome Issue of Science (265:1981f).Correlation between the location of the gene encoding the proteins ofthe invention on a physical chromosomal map and a specific disease orpredisposition to a specific disease, may help to delimit the region ofDNA associated with that genetic disease.

The nucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier or affectedindividuals. An analysis of polymorphisms, e.g. single nucleotidepolymorphisms may be carried out. Further, in situ hybridization ofchromosomal preparations and physical mapping techniques such as linkageanalysis using established chromosomal markers may be used for extendinggenetic maps. Often the placement of a gene on the chromosome of anothermammalian species, such as mouse, may reveal associated markers even ifthe number or arm of a particular human chromosome is not known. Newsequences can be assigned to chromosomal arms or parts thereof, byphysical mapping. This provides valuable information to investigatorssearching for disease genes using positional cloning or other genediscovery techniques. Once the disease or syndrome has been crudelylocalized by genetic linkage to a particular genomic region, forexample, AT to 11q22-23 (Gatti, R. A. et al. (1988) Nature 336:577-580), any sequences mapping to that area may represent associated orregulatory genes for further investigation. The nucleotide sequences ofthe subject invention may also be used to detect differences in thechromosomal location due to translocation, inversion, etc. among normal,carrier or affected individuals.

In another embodiment of the invention, the proteins of the invention,their catalytic or immunogenic fragments or oligopeptides thereof, an invitro model, a genetically altered cell or animal, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. One can identify effectors, e.g. receptors, enzymes,proteins, ligands, or substrates that bind to, modulate or mimic theaction of one or more of the proteins of the invention. The protein orfragment thereof employed in such screening may be free in solution,affixed to a solid support, borne on a cell surface, or locatedintracellulary. The formation of binding complexes, between the proteinsof the invention and the agent tested, may be measured. Agents couldalso, either directly or indirectly, influence the activity of theproteins of the invention.

In addition activity of the proteins of the invention against theirphysiological substrate(s) or derivatives thereof could be measured incell-based or cell-free assays. Agents may also interfere withposttranslational modifications of the protein, such as phosphorylationand dephosphorylation, farnesylation, palmitoylation, acetylation,alkylation, ubiquitination, proteolytic processing, subcellularlocalization and degradation. Moreover, agents could influence thedimerization or oligomerization of the proteins of the invention or, ina heterologous manner, of the proteins of the invention with otherproteins, for example, but not exclusively, docking proteins, enzymes,receptors, or translation factors. Agents could also act on the physicalinteraction of the proteins of this invention with other proteins, whichare required for protein function, for example, but not exclusively,their downstream signaling.

Methods for determining protein-protein Interaction are well known inthe art. For example binding of a fluorescently labeled peptide derivedfrom the interacting protein to the protein of the invention, or viceversa, could be detected by a change in polarisation. In case that bothbinding partners, which can be either the full length proteins as wellas one binding partner as the full length protein and the other justrepresented as a peptide are fluorescently labeled, binding could bedetected by fluorescence energy transfer (FRET) from one fluorophore tothe other. In addition, a variety of commercially available assayprinciples suitable for detection of protein-protein Interaction arewell known In the art, for example but not exclusively AlphaScreen(PerkinElmer) or Scintillation Proximity Assays (SPA) by Amersham.Alternatively, the interaction of the proteins of the invention withcellular proteins could be the basis for a cell-based screening assay,in which both proteins are fluorescently labeled and interaction of bothproteins is detected by analysing cotranslocation of both proteins witha cellular imaging reader, as has been developed for example, but notexclusively, by Cellomics or EvotecOAI. In all cases the two or morebinding partners can be different proteins with one being the protein ofthe invention, or in case of dimerization and/or oligomerization theprotein of the invention itself. Proteins of the invention, for whichone target mechanism of interest, but not the only one, would be suchprotein/protein interactions are PSAP, SFTPB, and/or FLJ40379.

Of particular interest are screening assays for agents that have a lowtoxicity for mammalian cells. The term “agent” as used herein describesany molecule, e.g. protein or pharmaceutical, with the capability ofaltering or mimicking the physiological function of one or more of theproteins of the invention. Candidate agents encompass numerous chemicalclasses, though typically they are organic molecules, preferably smallorganic compounds having a molecular weight of more than 50 and lessthan about 2,500 Daltons. Candidate agents comprise functional groupsnecessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise carbocyclic orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups.

Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acidsand derivatives, structural analogs or combinations thereof. Candidateagents are obtained from a wide variety of sources including librariesof synthetic or natural compounds. For example, numerous means areavailable for random and directed synthesis of a wide variety of organiccompounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. Where the screening assay is a binding assay, one ormore of the molecules may be joined to a label, where the label candirectly or indirectly provide a detectable signal.

Another technique for drug screening, which may be used, provides forhigh throughput screening of compounds having suitable binding affinityto the protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to the proteins of the inventionlarge numbers of different small test compounds, e.g. aptamers,peptides, low-molecular weight compounds etc., are provided orsynthesized on a solid substrate, such as plastic pins or some othersurface. The test compounds are reacted with the proteins or fragmentsthereof, and washed. Bound proteins are then detected by methods wellknown in the art. Purified proteins can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support. In another embodiment, onemay use competitive drug screening assays in which neutralizingantibodies capable of binding the protein specifically compete with atest compound for binding the protein. In this manner, the antibodiescan be used to detect the presence of any peptide, which shares one ormore antigenic determinants with the protein.

In a further embodiment, the present invention allows the production ofcells for the identification and/or characterisation of compounds whichstimulate beta-cell differentiation, insulin secretion and/or glucoseresponse, more particularly of compounds which increase the prosaposinexpression level or function. This method is particularly suitable forin vivo testing for diagnostic applications and drug development orscreening. The compound of interest is added to suitable cells and theprosaposin expression or function is determined. Alternatively, acompound of interest is added to a prosaposin-treated cell and theeffect on cell differentiation and/or insulin production is determined.Preferably, differentiated insulin-producing cells used. Insulin levelsin treated cells can be determined, e.g. quantified by Enzyme LinkedImmunoabsorbent Assay (ELISA) or Radio Immuno Assay (RIA). Using thismethod, a large number of compounds can be screened and compounds thatinduce prosaposin expression or support the activity of prosaposinleading to a beta-cell differentiation and/or an increase in insulinsecretion can be identified readily.

In a high-throughput screening method, the cells are transfected with aDNA construct, e.g. a viral or non-viral vector containing a reportergene, e.g. the lacZ gene or the GFP gene, under regulatory control of apromoter of a gene involved in beta-cell differentation, e.g. preferablya Pax4 promoter. The transfected cells are divided into aliquots andeach aliquot is contacted with a test substance, e.g. candidate 1,candidate 2, and candidate 3. The activity of the reporter genecorresponds to the capability of the test compound to induce beta-celldifferentiation.

In a further embodiment (which may be combined with the high-throughputscreening as described above) a medium throughput validation is carriedout. Therein, the test compound is added to cells being cultivated andthe prosaposin expression and/or the insulin production is determined.Following an initial high throughput assay, such as the cell based assayoutlined above where e.g. a Pax4 promoter is used as marker forbeta-cell regeneration, the activity of candidate molecules to inducebeta-cell differentiation is tested in a validation assay comprisingadding said compounds to the culture media of the embryoid bodies.Differentiation into insulin-producing cells is then evaluated, e.g. bycomparison to wild type and/or Pax4 expressing cells to assess theeffectiveness of a compound.

The nucleic acids encoding the protein of the invention can be used togenerate transgenic animals or site-specific gene modifications in celllines. These transgenic non-human animals are useful in the study of thefunction and regulation of the protein of the invention in vivo.Transgenic animals, particularly mammalian transgenic animals, can serveas a model system for the investigation of many developmental andcellular processes common to humans. A variety of non-human models ofmetabolic disorders can be used to test effectors/modulators of theprotein of the invention. Misexpression (for example, overexpression orlack of expression) of the protein of the invention, particular feedingconditions, and/or administration of biologically active compounds cancreate models of metablic disorders.

In one embodiment of the invention, such assays use mouse models ofinsulin resistance and/or diabetes, such as mice carrying gene knockoutsin the leptin pathway (for example, ob (leptin) or db (leptin receptor)mice).

Such mice develop typical symptoms of diabetes, show hepatic lipidaccumulation and frequently have increased plasma lipid levels (seeBruning J. C. et al., 1998, supra). Susceptible wild type mice (forexample C57BI/6) show similiar symptoms if fed a high fat diet. Inaddition to testing the expression of the proteins of the invention insuch mouse strains (see Examples section), these mice could be used totest whether administration of a candidate effector/modulator alters forexample lipid accumulation in the liver, in plasma, or adipose tissuesusing standard assays well known in the art, such as FPLC, colorimetricassays, blood glucose level tests, insulin tolerance tests and others.

Transgenic animals may be made through homologous recombination innon-human embryonic stem cells, where the normal locus of the geneencoding the protein of the invention is altered. Alternatively, anucleic acid construct encoding the protein of the invention is injectedinto oocytes and is randomly integrated into the genome. Vectors forstable integration include plasmids, retroviruses and other animalviruses, yeast artificial chromosomes (YACs), and the like. The modifiedcells or animals are useful in the study of the function and regulationof the protein of the invention. For example, a series of smalldeletions and/or substitutions may be made in the gene that encodes theprotein of the invention to determine the role of particular domains ofthe protein, functions in pancreatic differentiation, etc.

Furthermore, variants of the gene of the invention like specificconstructs of interest include anti-sense molecules, which will blockthe expression of the protein of the invention, or expression ofdominant negative mutations. A detectable marker, such as for examplelac-Z or luciferase may be introduced in the locus of the gene of theinvention, where up regulation of expression of the gene of theinvention will result in an easily detected change in phenotype.

One may also provide for expression of the gene of the invention orvariants thereof in cells or tissues where it is not normally expressedor at abnormal times of development. In addition, by providingexpression of the protein of the invention in cells in which they arenot normally produced, one can induce changes in cell behavior.

DNA constructs for homologous recombination will comprise at leastportions of the gene of the invention with the desired geneticmodification, and will include regions of homology to the target locus.DNA constructs for random integration do not need to contain regions ofhomology to mediate recombination. Conveniently, markers for positiveand negative selection are included. DNA constructs for randomintegration will consist of the nucleic acids encoding the protein ofthe invention, a regulatory element (promoter), an intron and apoly-adenylation signal. Methods for generating cells having targetedgene modifications through homologous recombination are known in theart. For non-human embryonic stem (ES) cells, an ES cell line may beemployed, or embryonic cells may be obtained freshly from a host, e.g.mouse, rat, guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer and are grown in the presence of leukemiainhibiting factor (LIF).

When non-human ES or embryonic cells or somatic pluripotent stem cellshave been transfected, they may be used to produce transgenic animals.After transfection, the cells are plated onto a feeder layer in anappropriate medium. Cells containing the construct may be selected byemploying a selective medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination or integration of the construct. Those colonies that arepositive may then be used for embryo transfection and morulaaggregation. Briefly, morulae are obtained from 4 to 6 week oldsuperovulated females, the Zona Pellucida is removed and the morulae areput into small depressions of a tissue culture dish. The ES cells aretrypsinized, and the modified cells are placed into the depressionclosely to the morulae. On the following day the aggregates aretransfered into the uterine horns of pseudopregnant females. Females arethen allowed to go to term. Chimeric offsprings can be readily detectedby a change in coat color and are subsequently screened for thetransmission of the mutation into the next generation (F1-generation).Offspring of the F1-generation are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogenic or congenic grafts or transplants, or in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimal, domestic animals, etc., for example, mouse, rat, guinea pig,sheep, cow, pig, and others. The transgenic animals may be used infunctional studies, drug screening, and other applications and areuseful in the study of the function and regulation of the protein of theinvention in vivo.

Finally, the invention also relates to a kit comprising at least one of

-   (a) a nucleic acid molecule coding for a protein of the invention or    a fragment thereof;-   (b) a protein of the invention or a fragment or an isoform thereof;-   (c) a vector comprising the nucleic acid of (a);-   (d) a host cell comprising the nucleic acid of (a) or the vector of    (c);-   (e) a polypeptide encoded by the nucleic acid of (a);-   (f) a fusion polypeptide encoded by the nucleic acid of (a);-   (g) an antibody, an aptamer or another modulator/effector of the    nucleic acid of (a) or the polypeptide of (b), (e) or (f) and-   (h) an anti-sense oligonucleotide of the nucleic acid of (a).

The kit may be used for diagnostic or therapeutic purposes or forscreening applications as described above. The kit may further containuser instructions.

The Figures show:

FIG. 1 shows the content of energy storage metabolites (ESM;triglyceride (TG) and glycogen) of Drosophila Saposin-related (GadFlyAccession Number CG12070) mutants. Shown is the change of triglyceridecontent of HD-EP(3) 36824 flies caused by integration of the P-vectorinto the annotated transcription unit (‘HD-36824 (TG, 70° C.)’, column 3and ‘HD-36824 (TG, 90° C.)’, column 6) in comparison to controlscontaining about 880 fly lines of the proprietary EP collection(‘HD-control (TG, 70° C.)’), column 1) and wild-type controls determinedin 4 independent assays (referred to as ‘WT-control (TG, 70° C.)’,column 2), and in comparison to controls containing about 2100 fly linesof the proprietary EP collection (‘HD-control (TG, 90° C.)’), column 4)and wild-type controls determined in more than 80 independent assays(referred to as ‘WT-control (TG, 90° C.)’, column 5). Also shown is thechange of glycogen content of HD-EP(3)36824 flies caused by integrationof the P-vector the into the annotated transcription unit (‘HD-36824(glycogen, 90° C.)’, column 8) in comparison to an internal assaycontrol including two wild-type strains and a one HD-line (referred toas ‘assay control (glycogen, 90° C.)’ column 7).

FIG. 2 shows the molecular organization of the mutated DrosophilaSaposin-related gene locus (referred to as Sap-r; GadFly AccessionNumber CG12070).

FIG. 3 shows human saposin-related proteins: Prosaposin (variant Gaucherdisease and variant metachromatic leukodystrophy, PSAP), humansurfactant, pulmonary-associated protein B (SFTPB), and humanhypothetical protein FLJ40379.

FIG. 3A shows the nucleic acid sequence of human PSAP (SEQ ID NO: 1).

FIG. 3B shows the amino acid sequence (one-letter code) of human PSAP(SEQ ID NO: 2).

FIG. 3C shows the nucleic acid sequence of human SFTPB (SEQ ID NO: 3).

FIG. 3D shows the amino acid sequence (one-letter code) of human SFTPB(SEQ ID NO: 4).

FIG. 3E shows the nucleic acid sequence of human FLJ40379 (SEQ ID NO:5).

FIG. 3F shows the amino acid sequence (one-letter code) of humanFLJ40379 (SEQ ID NO: 6).

FIG. 3G shows the comparison (ClustalW (1.83) protein sequence alignmentanalysis) of human and Drosophila proteins. Gaps in the alignment arerepresented as -. In the figure ‘PSAP Hs’ refers to human prosaposin,‘FLJ40379 Hs’ refers to human hypothetical protein FLJ40379, ‘SFTPB Hs’refers to human surfactant, pulmonary-associated protein B, and ‘Sap-rDm’ refers to Drosophila Saposin-related protein.

FIG. 4 shows the quantitative analysis of Sap-r homolog prosaposin(Psap) expression in mammalian tissues. The relative RNA-expression isshown on the Y-axis, in FIG. 4A to 4C the tissues tested are given onthe X-axis. WAT refers to white adipose tissue, BAT refers to brownadipose tissue. In FIG. 4D, the X-axis represents the time axis. ‘d0’refers to day 0 (start of the experiment), ‘d2’-‘d10’ refers to day2-day 10 of adipocyte differentiation).

FIG. 4A shows the quantitative analysis of Psap expression in mousewild-type tissues.

FIG. 4B shows the quantitative analysis of Psap expression in wild-typemice (wt-mice), compared to genetically obese mice (ob/ob-mice) and tofasted mice (fasted-mice).

FIG. 4C shows the quantitative analysis of Psap expression in mice fedwith a control diet compared to mice fed with a high fat diet.

FIG. 4D shows the quantitative analysis of Psap expression in mammalianfibroblast (3T3-L1) cells, during the differentiation from preadipocytesto mature adipocytes.

FIG. 5 shows the analysis of prosaposin (PSAP) expression in humanadipocytes.

FIG. 5A shows the expression of PSAP in human primary abdominaladipocyte cells, during the differentiation from preadipocytes to matureadipocytes.

FIG. 5B shows the expression of PSAP in human SGBS cells, during thedifferentiation from preadipocytes to mature adipocytes.

FIG. 6 shows the prosaposin dependent induction of the differentiationof insulin producing cells.

Mouse embryonic stem (ES) cells were differentiated as describedpreviously (patent application PCT/EP02/04362, published as WO02/086107, which is incorporated herein by reference). At the end of thedifferentiation procedure, cells were harvested and total RNA wasisolated. The abundance of insulin mRNA (FIG. 6A) and of beta-cellglucose transporter (Glut2) mRNA (FIG. 6B) was determined usingquantitative RT-PCR in an Applied Biosystems 7000 sequence detectiondevice. Levels were normalized using 18S RNA as control and a cyclenumber of 36 as reference. The numbers on the vertical line refer to theabundance of the indicated transcripts relative to an abundance forwhich 36 cycles are necessary for detection. ‘R1’ refers to unmodifiedmouse R1 embryonic stem (ES) cells; ‘Pax4’ refers to R1 mouse embryonicstem (ES) cells stably transfected with a CMV-Pax4 expression construct;‘insulin expression rel. to ACt36’ refers to expression of insulin inFIG. 6A and ‘Glut2 expression rel. to ACt36’ refers to expression ofbeta-cell glucose transporter Glut2 in FIG. 6B; ‘ES’ refers to mouseembryonic stem cells, as described in Example 7; ‘control 293 cells’refers to the differentiation protocol as described in Example 8, withthe addition of supernatant of 293 cells without prosaposin; ‘Psap’refers to the differentiation protocol as described in Example 9, withthe addition of prosaposin enriched supernatant of 293 cells todifferentiated cells).

The examples illustrate the invention:

EXAMPLE 1 Measurement of Energy Storage Metabolites (ESM) Contents inDrosophila

Mutant flies are obtained from a fly mutation stock collection. Theflies are grown under standard conditions known to those skilled in theart. In the course of the experiment, additional feedings with bakersyeast (Saccharomyces cerevisiae) are provided for the EP-lineHD-EP(3)36824. The average change of triglyceride and glycogen (hereinreferred to as energy storage metabolites, ESM) content of Drosophilacontaining the EP-vectors as homozygous viable integration wasinvestigated in comparison to control flies, respectively (see FIG. 1).For determination of ESM content, flies were incubated for 5 min at 70°C. or 90° C. in an aqueous buffer using a waterbath, followed by hotextraction. After another 5 min incubation at 70° C. or 90° C. and mildcentrifugation, the triglyceride content of the flies extract wasdetermined using Sigma Triglyceride (INT 336-10 or -20) assay bymeasuring changes in the optical density according to the manufacturer'sprotocol, and the glycogen content of the flies extract was determinedusing Roche (Starch UV-method Cat. No. 0207748) assay by measuringchanges in the optical density according to the manufacturer's protocol.As a reference the protein content of the same extract was measuredusing BIO-RAD DC Protein Assay according to the manufacturer's protocol.These experiments and assays were repeated several times.

The average triglyceride level of 883 fly lines of the proprietaryEP-collection determined at 70° C. (referred to as ‘HD-control (TG, 70°C.)’) is shown as 100% in the first column in FIG. 1. The averagetriglyceride level of Drosophila wild-type strain Oregon R fliesdetermined in 4 independent assays at 70° C. (referred to as ‘WT-control(TG, 70° C.)’) is shown as 116% in the second column in FIG. 1. Theaverage triglyceride level (μg triglyceride/μg protein) of 2108 flylines of the proprietary EP-collection determined at 90° C. (referred toas ‘HD-control (TG, 90° C.)’) is shown as 100% in the fourth column inFIG. 1. The average triglyceride level (μg triglyceride/μg protein) ofDrosophila wild-type strain Oregon R flies determined in 84 independentassays at 90° C. (referred to as ‘WT-control (TG, 90° C.)’) is shown as102% in the fifth column of FIG. 1. The average glycogen level (μgglycogen/μg protein) of an internal assay control consisting of twodifferent wild-type strains and an inconspicuous EP-line of the HD stockcollection (referred to as ‘assay control (glycogen, 90° C.)’) is shownas 100% in the seventh column in FIG. 1. The average triglyceride level(μg triglyceride/μg protein) of all flies of the EP collection (referredto as ‘EP-control’) is shown as 100% in the first column in FIG. 5.Standard deviations of the measurements are shown as thin bars.

HD-EP(3)36824 homozygous flies show constantly a lower triglyceridecontent than the controls (column 3 in FIG. 1, ‘HD-36824 (TG, 70° C.)’;column 6 in FIG. 1, ‘HD-36824 (TG, 90° C.)’). HD-EP(2)21554 homozygousflies also show a lower glycogen content than the controls (column 8 inFIG. 1, ‘HD-36824 (glycogen, 90° C.)’). Therefore, the loss of geneactivity is responsible for changes in the metabolism of the energystorage metabolites.

EXAMPLE 2 Identification of Drosophila Genes Responsible for Changes inMetabolite Contents

Genomic DNA sequences were isolated that are localized directly adjacentto the EP vector (herein HD-EP(3)36824) integration. Using the isolatedgenomic sequences from HD-EP(3)36824 homozygous flies public databaseslike Berkeley Drosophila Genome Project (GadFly) were screened. Thechromosomal localization site of HD-EP(3)36824 vector integration is atgene is locus 3R, 100A6-7 (Flybase and Gadfly). The homozygous viableintegration site of the HD-EP(3)36824 vector into base pair 535 of theSap-r transcript CG12070-RA and 61 base pairs 5prime of the Sap-rtranscript CG12070-RB in antisense orientation was confirmed. Therefore,expression of the cDNAs encoded by Sap-r could be affected byintegration of the vector of line HD-EP (3)36824 leading to a change inthe amount of energy storage metabolites. FIG. 2 shows the molecularorganization of this gene locus. A black double arrow in middle of theFigure represents the genomic DNA sequence. The space between two ticksrepresents a stretch of 1000 base pairs. The black triangle labeled‘HD-EP36824’ indicates the integration site of the EP-vector. Thetranscripts of Sap-r (as predicted by the Berkeley Drosophila GenomeProject) are shown as dark gray bars (exons) linked by dark gray lines(introns) in the lower half of the Figure labeled as ‘Sap-r’.

Table 1 is summarizing the data of our molecular analysis of theDrosophila proteins identified in this invention as being involved inthe regulation of the metabolism. TABLE 1 Molecular analysis ofDrosophila Saposin-related Analysis Result Protein domainsBeta-Ig-H3/Fasciclin (Flybase) InterPro Saposin type B, Saposin type A,Surfactant analysis protein B Locus 3R, 100A6-7 (Flybase, Gadfly release3) cDNA (sap-r) AI108030 (578 base pairs mRNA), AI109190 (635 base pairsmRNA) genomic DNA AE003775 RefSeq (Sap-r) NM_079858, NM_170529,NP_524597, NP_733408 Drosophila Homozygotes for deficiencies removingSap-r mutations & are fertile and viable and show no obvious mutantsphenotype. (Flybase)

EXAMPLE 3 Identification of the Human Saposin-Related HomologousProteins

Saposin-related homologous proteins and nucleic acid molecules codingtherefore are obtainable from insect or vertebrate species, e.g. mammalsor birds, preferably from humans, mouse, or Drosophila. Sequenceshomologous to Drosophila Saposin-related were identified using thepublicly available program BLASTP 2.2.3 of the non-redundant proteindata base of the National Center for Biotechnology Information (NCBI)(see, Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Table 2shows preferred human homologs of the Drosophila Saposin-related gene.

The term “polynucleotide comprising the nucleotide sequence as shown inGenBank Accession number” relates to the expressible gene of thenucleotide sequences deposited under the corresponding GenBank Accessionnumber. The term “GenBank Accession number” relates to NCBI GenBankdatabase entries (Ref.: Benson et al., Nucleic Acids Res. 28 (2000)15-18). TABLE 2 Human proteins homologous to Drosophila Saposin-relatedproteins I. Human proteins homologous to Drosophila Sap-r IA. Humanprosaposin (PSAP) NCBI human locus identification (ID): 5660, Hs PSAP,prosaposin (variant Gaucher disease and variant metachromaticleukodystrophy), 10q21-q22 RefSeq: GenBank Accession Number: NM_002778IB. Human surfactant, pulmonary-associated protein B (SFTPB) NCBI humanlocus identification (ID): 6439; Hs SFTPB, surfactant,pulmonary-associated protein B, 2p12-p11.2 Aliases: SP-B, PSP-B, SFTB3,SFTP3 RefSeq: GenBank Accession Number: NM_000542 IC. Human hypotheticalprotein FLJ40379 Nucleotide: ENSEMBL Accession Number: ENSG00000173005Protein: ENSEMBL Accession Number: ENSP00000308224, InternationalProtein Index (IPI) Accession Number: IPI00163920

EXAMPLE 4 Identification of Secreted Factors Expressed in Pancreas

A screen for secreted factors expressed in developing mouse pancreas wascarried out according to methods known by those skilled in the art (see,for example Pera E. M. and De Robertis E. M., (2000) Mech Dev 96(2):183-195) with several modifications.

Expression cDNA Library:

During organogenesis, the pancreatic bud is surrounded and influenced bythe associated mesenchyme. (see for example, Madsen O. D. et al., (1996)Eur. J. Biochem. 242: 435-445 and Slack, J. M., (1995) Development 121:1569-1580). Recently, it was suggested, that white adipocytes origindirectly from mesenchymal cells (Atanossova P. K., (2003) Folia Med. 45:41-45). During embryogenesis, the innervation and vascularization of thepancreas can be observed. Therefore, the tissue used in the screen mighthave contained besides pancreatic cells some adipocyte precursors, bloodvessels, as well as neuronal cells.

A mouse embryonic stage 9.5-15 pancreatic bud library was prepared inpCMVSPORT-6 vector using SUPERSCRIPT Plasmid System from Invitrogen(cat.#18248) according to the manufacturer's instructions. Thenon-amplified library was electroporated into MaxEff DH10B cells(Invitrogen).

Secretion Cloning

Bacterial clones from agar plates were picked with sterile toothpicksand cultured in 96-deep-well microtiter plates in 1 ml of LB-ampicillin(see Sambrook et al., supra). Aliquots of 8 cultures were pooled, andplasmid DNAs isolated by BioRobot_(—)9600 (QIAGEN) using QIAprep® TurboBioRobot Kit (QIAGEN). Human 293 cells were cultured in 75 ml tissueculture flasks in DMEM and 10% fetal calf serum. At 90-99% confluence,the cells were splitted at 1:3 ratio and plated at poly-D-lysin (Sigma)coated 96-well plates, 150 μl/well. Next day the cells were transfectedwith 100-500 ng plasmids using lipofectamine 2000 (Invitrogen), 0.5μl/well, in 100 μl/well Optimem (Invitrogen). After 6 h the medium wasexchanged for fresh complete growth medium. 24 hours after transfection,the cells were washed twice with DMEM w/o Cysteine and Methionine(Invitrogen), supplemented with 1% dialysed Bovine serum (Sigma) with 50mg/ml Heparin (Sigma) and glutamine. The cells were labeled in 50μl/well of the same medium and 0.75 μl/well (—S35 Met-label (HARTMANNANALYTIC GmbH, #44138). 12 hours later, 10 μl aliquots of thesupernatants were harvested in 96-well PCR plates and subjected to SDSgel electrophoresis in precast 420% gradient polyacrylamide Criteriongels (Biorad) under reducing conditions, using Criterion Dodeca Cell gelrunning chamber (Biorad). The gels were fixed in 10% acetic acid, 25%isopropanol for 30 min, soaked 15-30 min in AMPLIFY reagent (Amersham),dried and exposed to X-OMAT (AR) film (Kodak). Positive clones wereidentified by sub-selection. The 8 individual bacterial clones of eachpositive pool were regrown in 96-well-plates, DNA of individual cloneswas prepared and used for transfection as described. If one of theclones yielded proteins of the same size as that of the original pool, apositive clone was identified. Positive clones were partially sequencedfrom the 5′ end (SEQLAB, Goettingen). Sequences (of about 500nucleotides) were compared to public nucleotide and protein databases toreveal similarity to previously described proteins.

EXAMPLE 5 Expression of the Polypeptides in Mammalian Tissues

To analyse the expression of the polypeptides disclosed in thisinvention in mammalian tissues, several mouse strains (preferably micestrains C57BI/6J, C57BI/6 ob/ob and C57BI/KS db/db which are standardmodel systems in obesity and diabetes research) were purchased fromHarlan Winkelmann (33178 Borchen, Germany) and maintained under constanttemperature (preferably 22° C.), 40 percent humidity and a light/darkcycle of preferably 14/10 hours. The mice were fed a standard chow (forexample, from ssniff Spezialitäten GmbH, order number ssniff M-ZV1126-000). For the fasting experiment (“fasted wild-type mice”),wild-type mice were starved for 48 h without food, but only watersupplied ad libitum (see, for example, Schnetzler et al. (1993) J ClinInvest 92: 272-280, Mizuno et al., (1996) Proc Natl Acad Sci USA 93:3434-3438). Animals were sacrificed at an age of 6 to 8 weeks. Theanimal tissues were isolated according to standard procedures known tothose skilled in the art, snap frozen in liquid nitrogen and stored at−80° C. until needed.

For analyzing the role of the proteins disclosed in this invention inthe in vitro differentiation of different mammalian cell culture cellsfor the conversion of pre-adipocytes to adipocytes, mammalian fibroblast(3T3-L1) cells (e.g., Green & Kehinde, Cell 1: 113-116, 1974) wereobtained from the American Tissue Culture Collection (ATCC, Hanassas,Va., USA; ATCC-CL 173). 3T3-L1 cells were maintained as fibroblasts anddifferentiated into adipocytes as described in the prior art (e.g., Qiu.et al., J. Biol. Chem. 276: 11988-11995, 2001; Slieker et al., BBRC 251:225-229, 1998). In brief, cells were plated in DMEM/10% FCS (Invitrogen,Karlsruhe, Germany) at 50,000 cells/well in duplicates in 6-well plasticdishes and cultured in a humidified atmosphere of 5% CO₂ at 37° C. Atconfluence (defined as day 0: d0) cells were transferred to serum-free(SF) medium, containing DMEM/HamF12 (3:1; Invitrogen), fetuin (300μg/ml; Sigma, Munich, Germany), transferrin (2 μg/ml; Sigma),pantothenate (17 μM; Sigma), biotin (1 μM; Sigma), and EGF (0.8 nM;Hoffmann-La Roche, Basel, Switzerland). Differentiation was induced byadding dexamethasone (DEX; 1 μM; Sigma),3-methyl-isobutyl-1-methylxanthine (MIX; 0.5 mM; Sigma), and bovineinsulin (5 μg/ml; Invitrogen). Four days after confluence (d4), cellswere kept in SF medium, containing bovine insulin (5 μg/ml) untildifferentiation was completed. At various time points of thedifferentiation procedure, beginning with day 0 (day of confluence) andday 2 (hormone addition; for example, dexamethasone and3-isobutyl-1-methylxanthine), up to 10 days of differentiation, suitablealiquots of cells were taken every two days.

RNA was isolated from mouse tissues or cell culture cells using TrizolReagent (for example, from Invitrogen, Karlsruhe, Germany) and furtherpurified with the RNeasy Kit (for example, from Qiagen, Germany) incombination with an DNase-treatment according to the instructions of themanufacturers and as known to those skilled in the art. Total RNA wasreverse transcribed (preferably using Superscript II RNaseH—ReverseTranscriptase, from Invitrogen, Karlsruhe, Germany) and subjected toTaqman analysis preferably using the Taqman 2xPCR Master Mix (fromApplied Biosystems, Weiterstadt, Germany; the Mix contains according tothe Manufacturer for example AmpliTaq Gold DNA Polymerase, AmpErase UNG,dNTPs with dUTP, passive reference Rox and optimized buffer components)on a GeneAmp 5700 Sequence Detection System (from Applied Biosystems,Weiterstadt, Germany).

EXAMPLE 6 Analysis of the Differential Expression of Transcripts of theProteins of the Invention in Human Tissues

RNA preparation from human primary adipose tissues and a human adipocytecell line (SGBS) was done as described in Example 5. The targetpreparation, hybridization, and scanning was performed as described inthe manufactures manual (see Affymetrix Technical Manual, 2002, obtainedfrom Affmetrix, Santa Clara, USA).

The expression analysis (using Affymetrix GeneChips) of the prosaposin(PSAP) gene using primary human abdominal adipocycte and SGBS celldifferentiation clearly shows differential expression of human PSAP inadipocytes. Several independent experiments were done. All experimentsshow that the PSAP transcripts are the most abundant at day 12 comparedto day 0 during differentiation. These data further confirm the mouse3T3L-1 differentiation data.

Thus, the PSAP protein has to be significantly increased in order forthe preadipocyctes to differentiate into mature adipocyctes. The PSAPprotein in preadipocyctes has the potential to enhance adiposedifferentiation. Therefore, the PSAP protein might play an essentialrole in the regulation of human metabolism, in particular in theregulation of adipogenesis and thus it might play an essential role inobesity, diabetes, and/or metabolic syndrome.

EXAMPLE 7 Generation of ES Cells Expressing the Pax4 Gene

Mouse R1 ES cells (Nagy et al., (1993) Proc. Natl. Acad. Sci. USA 90:8424-8428) were electroporated with the Pax4 gene under the control ofthe CMV promoter and the neomycin resistance gene under the control ofthe phosphoglycerate kinase I promoter (pGK-1).

ES cells were cultured in Dulbecco's modified Eagle's medium containing4.5 g/l glucose, 10-4 M beta-mercaptoethanol, 2 nM glutamine, 1%non-essential amino acids, 1 nM Na-pyruvate, 20% FCS and 500 U/mlleukaemia inhibitory factor (LIF). Briefly, approximately 10⁷ ES cellsresuspended in 0.8 ml phosphate buffered saline (PBS) were subjected toelectroporation with 25 □g/ml of linearized expression vector (Joyner,Gene Targeting: A Practical Approach, Oxford University Press, New York,1993). Five minutes after electroporation, ES cells were plated on petridishes containing fibroblastic feeder cells previously inactivated bytreatment with 100 μg/ml mitomycin C. One day after electroporation,culture medium was changed to medium containing 450 μg/ml G418.Resistant clones were separately isolated and cultured 14 days afterapplying the selection medium. Cells were always cultured at 37° C., 5%CO₂. These untreated and undifferentiated ES cells were used as controlthe experiment shown in FIG. 6 (refered to as ‘ES’ in FIG. 6).

EXAMPLE 8 Differentiation of ES Cells into Insulin-Producing Cells(Refered to as ‘Control 293 Cells’ in FIG. 6)

The ES cell line R1 (wild type, ‘R1’ in FIG. 6) and ES cellsconstitutively expressing Pax4 (‘Pax4’ in FIG. 6) were cultivated asembryoid bodies (EB) by the hanging drop method, as described in patentapplication PCT/EP02/04362, published as WO 02/086107, which isincorporated herein by reference, with media as described below and inTable 3. The embryoid bodies were allowed to form in hanging dropcultures for 2 days and then transferred for three days to suspensioncultures in petri dishes. At day 5, EBs were plated separately ontogelatin-coated 6 cm cell culture dishes containing a differentiationmedium prepared with a base of Iscove modified Dulbecco's medium. Afterdissociation and replating at day 14 cells were cultured up to 40 daysin the differentiation medium prepared with a base of Dulbecco'smodified Eagle's medium: Nutrient Mixture F-12 (DMEM/F12).

EXAMPLE 9 Expression of Pancreas Specific Genes After Differentiation ofES Cells into Insulin-Producing Cells

Expression levels of pancreas specific genes was measured by Taqmananalysis as described in Example 5. Total RNA was isolated fromundifferentiated R1 and Pax4⁺ ES cells (control ES cells) at day 0 anddifferentiated R1 ES and Pax4⁺ ES cells at day 40. RNA preparation wasdone as described in Example 5 without using Trizol reagent.

Results show that markers for beta-cell differentiation function wereexpressed at higher levels in Pax4⁺ differentiated ES cells than indifferentiated wild type ES cells demonstrating that activation of apancreatic developmental control gene renders differentiation moreefficient than for wild type ES cells (FIG. 6). Expression of Glut2 indifferentiated stem cells indicates that hormone-producing cells arecapable of responding to glucose. Expression of substantial amounts ofinsulin in differentiated stem cells indicates that differentiated cellsshow a phenotype similar to beta-cells.

EXAMPLE 10 Induction of Differentiation of Insulin-Producing Cells byProsaposin (Refered to as ‘Psap’ in FIG. 6)

In order to study the effect of prosaposin to induce beta-celldifferentiation in vitro, stable mouse embryonic stem (ES) cellsexpressing the Pax4 under the control of the cytomegalovirus (CMV) earlypromoter/enhancer region were generated as described in Example 7. Pax4and wild type ES cells were then cultured in hanging drops or spinnercultures to allow the formation of embryoid bodies. Embryoid bodies weresubsequently plated, enzymatically dissociated, and replated. Afterdissociation, cells were cultured in a differentiation medium containingvarious growth factors (see Table 3 for more detail). Additionallyprosaposin enriched supernatant of 293 cells was added every second dayuntil day 40. Under such conditions, the expression of insulin wassignificantly induced by prosaposin (FIG. 6A). In addition, the additionof prosaposin to the differentiation medium did enhance the expressionof the glucose transporter Glut2 (FIG. 6B). By comparison, wild type EScells did contain only very small numbers of insulin- and Glut2producing cells at the same stage. These data demonstrate thatprosaposin can significantly promote and enhance ES cellsdifferentiation into insulin-producing cells compared to wild type EScells.

The results shown in FIG. 6 clearly demonstrate a significant inductionof the differentiation of insulin-producing (FIG. 6A) and glucoseresponsive (FIG. 6B) cells, if prosaposin is added on later stages ofdifferentiation. Thus, prosaposin has a strong inductive effect on thedifferentiation of insulin-producing beta cells. TABLE 3 Protocol forthe induction of differentiation of insulin-producing cells byprosaposin (Psap) Media B2 and B27 are described in Rolletschek et al.,(2001) Mech. Dev. 105: 93-104. Stage of Coating and Day CultivationMedium Analysis 0 hanging drops Iscove + 20% FCS RNA (ES cells) (600cells/drop) 1 2 EBs in suspension Iscove + 20% FCS 3 4 5 plating of EBsIscove + 20% FCS gelatin coating +1 ornithine/ +2 laminin coating +3 +4+5 +6 +7 +8 +9 dissociation B2 + B27 + NA + 10% FCS RNA (1 × 6 cm dish)+10 medium change B2 + B27 + NA + Psap, 50 ng/ml +11 +12 medium change+Psap, 50 ng/ml +13 +14 medium change +Psap, 50 ng/ml +15 +16 mediumchange +Psap, 50 ng/ml +17 +18 medium change +Psap, 50 ng/ml +19 +20medium change +Psap, 50 ng/ml +21 +22 medium change +Psap, 50 ng/ml +23+24 medium change +Psap, 50 ng/ml +25 +26 medium change +Psap, 50 ng/ml+27 +28 medium change +Psap, 50 ng/ml +29 +30 medium change +Psap, 50ng/ml +31 +32 medium change +Psap, 50 ng/ml RNA

EXAMPLE 11 Functional Characterisation of the DifferentiatedInsulin-Producing Cells

One important property of beta-cells is glucose responsive insulinsecretion. To test whether the Pax4 derived insulin-producing cellspossessed this glucose responsive property, an in vitro glucoseresponsive assay can be performed on the differentiated cells. On theday of the assay, the differentiation medium of 12 or 6 well plate isremoved and the cells are washed 3 times with Krebs Ringer BicarbonateHepes Buffer (K#RBH; 125 mM NaCl, 4.74 mM KCl, 1 mM CaCl₂, 1.2 mMKH₂PO₄, 1.2 mM MgSO₄, 5 mM NaHCO₃, 25 mM Hepes, pH 714 and 0.1% BSA)supplemented with 2.8 mM Glucose. For Preincubation cells were incubatedin KRBH+2.8 mM Glucose for 2 hours at 37° C. Afterwards cells wereincubated in 500 ml KRBH+2.8 mM Glucose for 1 hour and the supernatantis then kept for measurement of basal insulin secretion. For thestimulated insulin release 500 ml KRBH containing 27.7 mM glucose isadded to the cells. After 1 hour incubation at 37° C., the KRBH isrecovered for measurement of glucose-induced insulin secretion and thecells were extracted with acid-ethanol. (see also Irminger, J.-C. etal., 2003, Endocrinology 144: 1368-1379). Insulin levels can bedetermined by an Enzyme-Linked Immunosorbent Assay (ELISA) for mouseinsulin (Mercodia) and performed according to the manufacturer'srecommendations.

EXAMPLE 12 Transplantation of Pax4 ES Derived Insulin-Producing Cells inSTZ Diabetic Mice

The therapeutic potential of prosaposin induced insulin-producing cellsto improve and cure diabetes can be investigated by transplanting thecells into streptozotocin induced diabetic mice. Streptozotocin is anantibiotic which is cytotoxic to beta-cells when administered at certaindosage (see Rodrigues et al.: Streptozotocin-induced diabetes, inMcNeill (ed) Experimental Models of Diabetes, CRC Press LLC, 1999). Itseffect is rapid, rendering an animal severely diabetic within 48 hours.

Non-fasted Male BalbC mice can be treated with STZ to develophyperglycaemia after STZ treatment. Mice are considered diabetic if theyhave a blood glucose level above 10 mmol/l for more than 3 consecutivedays. Cells are transplanted under the kidney capsule and into thespleen of animals. The presence of the insulin-producing cells can beconfirmed by immunohistological analysis of the transplanted tissue.Results are expected to demonstrate that the transplanted cells cannormalise blood glucose in diabetic animals.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1-36. (canceled)
 37. The use of a saposin-related product and/or amodulator/effector thereof to promote the protection, survival and/orregeneration of insulin producing cells comprising administering to thecells of a patient in need thereof an effective amount of asaposin-related product and/or a modulator/effector thereof.
 38. The useof claim 37, wherein the insulin producing cells are beta-cells.
 39. Theuse of claim 37, wherein the insulin producing cells are of mammalianorigin, preferably of human origin.
 40. The use of claim 37, wherein theinsulin producing cells have been transfected with a pancreatic gene,particularly the Pax4 gene.
 41. The use of claim 37 for the preventionor treatment of a disease going along with impaired beta-cell function,particularly for the treatment of beta-cell degeneration in patientssuffering from diabetes type I, LADA, or progressed diabetes type II, orfor the prevention of beta-cell degeneration in patients at risk todevelop beta-cell degeneration, like for example but not limited topatients suffering from diabetes type I or II, or LADA in early stages.42. The use of claim 37, wherein a saposin-related product or amodulator/effector thereof that influences the expression level orfunction of a saposin-related product is administered to a patient (i)as a pharmaceutical composition e.g. enterally, parenterally ortopically directly to the pancreas, (ii) via implantation ofsaposin-related protein product expressing cells, and/or (iii) via genetherapy.
 43. The use of claim 42, wherein the saposin-related product ormodulator/effector thereof is administered in combination with anotherpharmaceutical composition useful to treat beta-cell degeneration, forexample but not limited to hormones, growth factors, or immunemodulating agents.
 44. The use of claim 37, wherein the saposin-relatedproduct is a protein including purified natural, synthetic orrecombinant saposin-related products and variants thereof.
 45. The useof claim 44, wherein the saposin-related product is of mammalian origin,preferably human origin, more preferably selected from proteins orpeptides substantially homologous to the human saposin-related precursorproteins as shown in Table
 2. 46. The use of claim 37, wherein thesaposin-related product is a nucleic acid, e.g. RNA and/or DNA encodinga saposin-related protein product.
 47. The use of claim 37, wherein thedifferentiation of progenitor, e.g. stem cells into insulin-producingcells in vitro comprises a) optionally activating one or more pancreaticgenes in progenitor cells, b) optionally aggregating said cells to formembryoid bodies, c) cultivating said cells or embryoid bodies inspecific differentiation media containing saposin-related proteinproduct and d) identifying and optionally selecting insulin-producingcells.
 48. The use of claim 47, wherein the saposin-related treatedinsulin producing cells are (i) capable of a response to glucose and/or(ii) capable of expressing glucagon.
 49. The use of claim 47, whereinthe saposin-related insulin producing cells are capable of normalizingblood glucose levels after transplantation into mice.
 50. The use ofclaim 37, wherein an effective amount of in vitro saposin-related cellsare transplanted to a patient in need.
 51. The use of claim 37,comprising a stimulation of saposin-related expression, wherein cellsfrom a patient in need that have been modified to produce and secrete asaposin-related protein product in vitro are re-implanted into thepatient and/or wherein cells of a patient in need are modified toproduce and secrete a saposin-related protein product in vivo.
 52. Amethod for differentiating or regenerating cells into functionalpancreatic cells, the method comprising: (a) cultivating cells capableof being differentiated or regenerated into pancreatic cells in thepresence of an effective amount of a saposin-related protein in vitro(b) allowing the cells to develop, to differentiate and/or to regenerateat least one pancreatic function; and (c) optionally preparing aneffective amount of the differentiated or regenerated pancreatic cellsfor transplantation into a patient in need thereof, particularly a humanindividual.
 53. The method of claim 52, wherein the patient in need has(a) functionally impaired, (b) reduced numbers and/or (c) functionallyimpaired and reduced numbers of pancreatic cells.
 54. The method ofclaim 52, wherein said patient in need is a type I diabetic patient ortype II diabetic patient or LADA patient.
 55. The method of claim 52,wherein the pancreatic cells are insulin-producing cells.
 56. The methodof claim 52, wherein the pancreatic cells are beta-cells of thepancreatic islets.
 57. The method of claim 52, wherein the cells in step(a) are selected from embryonic stem cells, adult stem cells, or somaticstem cells.
 58. The method of claim 52, wherein the cells in step (a)are of mammalian origin, preferably human origin.
 59. The method ofclaim 52, wherein the protein is added at concentrations between 1 ng/mland 500 ng/ml, preferably between 10 and 100 ng/ml, more preferably atabout 50 ng/ml.
 60. The method of claim 52, wherein the at least onepancreatic function is selected from insulin production in response toglucose and expression of glucagon.
 61. A method for differentiating orregenerating cells into functional pancreatic cells, the methodcomprising: preparing an effective amount of a saposin-related productor of cells capable of expressing a saposin-related product foradministration to a patient in need thereof.
 62. The method of claim 61,wherein the saposin-related product is a protein or a nucleic acid. 63.The method of claim 61, wherein cells have been modified to produce andsecrete a saposin-related protein product and are prepared fortransplantation into a suitable location in the patient. 64-82.(canceled)